Methods of use of anti-cd19/anti-cd38 common light chain bispecific antibodies

ABSTRACT

Provided are anti-CD19 and anti-CD38 common light chain bispecific antibodies. The anti-CD-19 and anti-CD38 bispecific antibodies described herein are useful in methods for treating a cancer or a tumor.

CROSS-REFERENCE

This application is a Divisional of U.S. patent application Ser. No.17/229,751, filed Apr. 13, 2021, which is a Continuation ofInternational Application No. PCT/US2021/019685, filed on Feb. 25, 2021,which claims the benefit of U.S. Provisional Application No. 62/981,990filed on Feb. 26, 2020, U.S. Provisional Application No. 62/990,330filed on Mar. 16, 2020, and U.S. Provisional Application No. 63/094,838filed on Oct. 21, 2020, which applications are incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 5, 2021, isnamed 51527-712_403_SL.txt and is 104,266 bytes in size.

BACKGROUND

Antibody therapeutics have been used successfully to treat a variety ofdiseases; however, their application can be limited with respect toclinical efficacy in complex diseases such as cancer. Engineeringantibody-based therapeutics to alter target-binding affinities andvalences provides a potential pathway towards achieving increasedefficacy and improving treatment outcomes. Bispecific or multivalentantibodies thus offer a potential approach to resolving challenges tiedto the multifactorial nature of complex diseases. By binding twodifferent antigenic molecules or different epitopes of the same antigen,bispecific antibodies offer greater functionality and offer a widevariety of applications as targeting agents for the treatment of anumber of diseases.

SUMMARY

The dynamic relationship between cancer biology and the immune system isa factor associated with clinical outcomes. The immune response plays asignificant role in regulating the tumor microenvironment during cancerdevelopment. Immune cells such as T cells and B cells thus act asmodulators and effectors of cancer progression or metastasis. Notably,immunosuppressive cells play an important role in the anti-tumor immuneresponse wherein immunosuppression is generally associated with tumorgrowth and invasion, and correlates with negative outcomes. Although Bcells are known to positively modulate the immune response, populationsof immunosuppressive B cells function to suppress the anti-tumor immuneresponse thus facilitating tumor growth.

Provided herein are certain binding molecules that targetimmunosuppressive B-cell populations with bispecific or multivalenttargeting molecules. Targeting immune suppressive B-cell populationspresents a pathway for therapeutic intervention in cancer thateffectively modulates the anti-tumor immune response to improvetreatment outcomes (e.g. in contrast to selective depletion of anepithelial cancer cell population). The binding molecules providedherein can comprise a bispecific antibody that binds to a B-cell lineagesurface marker (e.g., CD19, CD138, IgA, and/or CD20) and a surfacemarker of immunosuppressive B cells (e.g., IgD, CD1, CD5, CD21, CD24,CD38, HM13, SLAMF7, AQP3, and/or latent TGF-beta (e.g., TGF-beta LAP)).In a certain specific embodiment, the bispecific antibody binds to CD19and CD38, thus possessing selectivity for a specific immunosuppressiveB-cell population.

As provided and described herein, the bispecific antibodies that bind toCD19 and CD38 provide advantages in the selective binding of cellsexpressing CD19 and CD38 (e.g. immunosuppressive B-cell populations).Furthermore, the bispecific antibodies, disclosed herein, that bind toCD19 and CD38 demonstrate advantages in that they do not promotehemolysis or hemagglutination, especially when compared to mono-specificCD19 or CD38 antibodies. Thus, overcoming severe side-effects seen withmonospecific CD19 or CD38 antibodies (e.g., SARCLISA®(isatuximab-irfc)), such as anemia. Bispecific antibodies that bind toCD19 and CD38 also demonstrate advantages in that they effectivelypromote advantageous target cell apoptosis of cells expressing CD19 andCD38, especially when compared to mono-specific controls. Furthermore,the bispecific antibodies that bind to CD19 and CD38 further provideadvantages over the mere use of two independent monoclonal antibodiesindependently targeting CD38 and CD19, in that they more effectivelytarget specific immunosuppressive B-cell populations, leading to greatereffectiveness and potentially lower side effects seen with other B celltargeting monoclonal antibodies (e.g., Rituximab), such as orlymphopenia.

Described herein is a composite binding molecule comprising a firstbinding component configured to bind a first target and a second bindingcomponent configured to bind a second target, wherein the first targetcomprises a B-cell lineage surface marker, and wherein the second targetcomprises an immunosuppressive B-cell surface marker, wherein the firsttarget and the second target are not identical. In some embodiments, thefirst or the second binding component comprises a polypeptide. In someembodiments, the first or the second binding component consists of apolypeptide. In some embodiments, the first and the second bindingcomponent comprise a polypeptide. In some embodiments, the first and thesecond binding component consist of a polypeptide. In some embodiments,the polypeptide of the first or second binding component comprises anamino acid sequence at least 100 amino acid residues in length. In someembodiments, the polypeptide of the first and second binding componentcomprise an amino acid sequence at least 100 amino acid residues inlength.

In some embodiments, the B-cell lineage surface marker comprises CD19,CD138, IgA, or CD45. In some embodiments, the B-cell lineage surfacemarker comprises CD19. In some embodiments, the B-cell lineage surfacemarker consists of CD19. In some embodiments, the immunosuppressiveB-cell surface marker comprises IgD, CD1, CD5, CD21, CD24, CD38, HM13,SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP). In someembodiments, the immunosuppressive B-cell surface marker comprises CD38.In some embodiments, the immunosuppressive B-cell surface markerconsists of CD38.

In some embodiments, the first or second binding component comprise animmunoglobulin heavy and light chain pair, an scFv, a F(ab), a F(ab′)₂,a single domain antibody, a variable region fragment from animmunoglobulin new antigen receptor (V_(NAR)), or a variable regionderived from a heavy chain antibody (V_(H)H). In some embodiments, thefirst and second binding component comprise an immunoglobulin heavy andlight chain pair, an scFv, a F(ab), a F(ab′)₂, a single domain antibody,a variable region fragment from an immunoglobulin new antigen receptor(V_(NAR)), or a variable region derived from a heavy chain antibody(V_(H)H).

In some embodiments, the first or second binding component comprises animmunoglobulin heavy and light chain pair. In some embodiments, thefirst and second binding component comprise an immunoglobulin heavy andlight chain pair. In some embodiments, the composite binding moleculecomprises an immunoglobulin heavy chain and an immunoglobulin lightchain, wherein the immunoglobulin heavy chain comprises an HCDR1 aminoacid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2 aminoacid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155, anHCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91-95; andan immunoglobulin light chain comprises an LCDR1 amino acid sequence setforth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence setforth in any one of SEQ ID NOs: 51-55, and/or an LCDR3 amino acidsequence set forth in any one of SEQ ID NOs: 61-65. In some embodiments,the immunoglobulin heavy chain comprises an amino acid sequence havingat least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 3; and theimmunoglobulin light chain having at least about 90%, 95%, 97%, 99%identity to SEQ ID NO: 2. In some embodiments, the immunoglobulin heavychain comprises an amino acid sequence identical to that set forth inSEQ ID NO: 3; and the immunoglobulin light chain comprises an amino acidsequence identical to that set forth in SEQ ID NO: 2. In someembodiments, composite binding molecule is a common light chainbispecific IgG.

In some embodiments, the first or second binding component comprises anscFv. In some embodiments, the first and second binding componentcomprise an scFv. In some embodiments, the composite binding molecule isa bispecific antibody or dual-antigen binding fragment thereof.

In some embodiments, the bispecific antibody is selected from one of thefollowing formats: a common light chain bispecific IgG, a Fab-Fc:scFv-Fcbispecific IgG, a Fab-Fc-Fab:Fc bispecific IgG, aFab-Fc-scFv:Fab-Fc-scFv bispecific IgG, a Fab-Fc-scFv:Fc bispecific IgG,a Fab-Fc-Fab:Fab-Fc bispecific IgG, an scFv-Fab-Fc:scFv-Fab-Fcbispecific IgG, a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG, aFab-Fc-Fab:Fab-Fc-Fab bispecific IgG, scFv-Fab-Fc:Fc bispecific IgG, anda Fab-Fc-scFv:Fab-Fc bispecific IgG. In some embodiments, the bispecificantibody is a Fab-Fc:scFv-Fc bispecific IgG. In some embodiments, thebispecific antibody is a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG. In someembodiments, the bispecific antibody is an scFv-Fab-Fc:Fc bispecificIgG. In some embodiments, the composite binding molecule comprises an Fcregion comprising a native carbohydrate or an afucosylated carbohydratemodified amino acid residue. In some embodiments, the nativecarbohydrate or the afucosylated carbohydrate modified amino acidresidue corresponds to Asparagine 297 according to EU numbering.

In some embodiments, the first binding component comprises an HCDR1amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2amino acid sequence set forth in any one of SEQ ID NOs: 21-25, an HCDR3amino acid sequence set forth in any one of SEQ ID NOs: 31-35, an LCDR1amino acid sequence set forth in any one of SEQ ID NOs: 41-45, an LCDR2amino acid sequence set forth in any one of SEQ ID NOs: 51-55, and/or anLCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61-65.

In some embodiments, the first binding component comprises an amino acidsequence comprising at least about 90%, 95%, 97%, 99% identity to, or is100% identical to the amino acid sequences set forth in any one of SEQID NOs: SEQ ID NO: 1 and SEQ ID NO: 2.

In some embodiments, the first binding component comprises an amino acidsequence identical to the amino acid sequences set forth in SEQ ID NO: 1and SEQ ID NO: 2.

In some embodiments, the second binding component comprises an HCDR1amino acid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or150-155, an HCDR3 amino acid sequence set forth in any one of SEQ IDNOs: 91-95, an LCDR1 amino acid sequence set forth in any one of SEQ IDNOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQID NOs: 111-115, and/or an LCDR3 amino acid sequence set forth in anyone of SEQ ID NOs: 121-125.

In some embodiments, the second binding component comprises an aminoacid sequence comprising at least about 90%, 95%, 97%, 99% identity to,or is 100% identical to the amino acid sequences set forth in any one ofSEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, the second bindingcomponent comprises an amino acid sequence identical to the amino acidsequences set forth in SEQ ID NO: SEQ ID NO: 3 and SEQ ID NO: 4.

In some embodiments, the composite binding molecule binds to CD19+,CD38+B cells.

Disclosed is a cell comprising the nucleic acid encoding a compositebinding molecule. In some embodiments, the polynucleotide sequenceencoding the composite binding molecule is operatively coupled to aeukaryotic regulatory sequence. In some embodiments, the cell comprisesa prokaryotic cell. In some embodiments, the prokaryotic cell is anEscherichia coli cell. In some embodiments, the cell comprises aeukaryotic cell. In some embodiments, the eukaryotic cell is a ChineseHamster Ovary (CHO) cell, an NS0 murine myeloma cell, or a human PER.C6cell.

Disclosed is a composition comprising a composite binding molecule and apharmaceutically acceptable diluent, carrier, or excipient. In someembodiments, the composition is formulated for intravenousadministration. In some embodiments, the composition is formulated forsubcutaneous administration.

Provided are composite binding molecules for use in methods of treatinga tumor or a cancer in an individual. In some embodiments, the cancer orthe tumor is a hematologic cancer. In some embodiments, thehematological cancer is a B cell malignancy. In certain embodiments, theB cell malignancy is B-cell Acute Lymphocytic Leukemia. In certainembodiments, the B cell malignancy is Chronic Lymphocytic Leukemia,Small Lymphocytic Lymphoma, Mantle Cell Lymphoma, or Non-HodgkinsLymphomas (Diffuse Large B-cell Lymphoma, Follicular Lymphoma). In someembodiments, the hematological cancer is a plasma malignancy. In certainembodiments, the plasma malignancy is multiple myeloma. In someembodiments of any of the preceding embodiments, the hematologicalcancer expresses CD19 and CD38 (e.g. cells of the cancer express CD19and CD38).

In some embodiments, the cancer or the tumor is a solid-tissue cancer.In some embodiments, the cancer comprises breast cancer, prostatecancer, pancreatic cancer, lung cancer, kidney cancer, stomach cancer,esophageal cancer, skin cancer, colorectal cancer, brain cancer, or headand neck cancer. In some embodiments, the breast cancer is triplenegative breast cancer, the lung cancer is non-small cell lung cancer,the head and neck cancer is head and neck squamous cell cancer, thekidney cancer is renal cell carcinoma, the brain cancer is glioblastomamultiforme, or the skin cancer is melanoma.

Provided are composite binding molecules for use in a method of reducingimmunosuppressive B cells in, adjacent to, or surrounding a tumor of anindividual or immunosuppressive B cells affecting an anti-tumor immuneresponse of an individual that are distant from the tumor site. Providedare composite binding molecules for use in a method of reducingimmunosuppressive B cells in, adjacent to, or surrounding a tumor of anindividual. In some embodiments, the tumor infiltrating B cells or theimmunosuppressive B cells comprise CD19+ CD38+ B cells. Further providedare composite binding molecules for use in a method of reducing orinhibiting the function of immunosuppressive B cells in, adjacent to, orsurrounding a tumor of an individual and/or immunosuppressive B cellsaffecting an anti-tumor immune response of an individual that aredistant from a tumor site. In some embodiments, the function ofimmunosuppressive B cells comprises the release of anti-inflammatory orimmunosuppressive cytokines such as IL-10, IL 35, TGF-beta, or acombination thereof

Disclosed are methods of treating an individual afflicted with a canceror a tumor comprising administering to the individual afflicted with thecancer or the tumor the composite binding molecule, thereby treating thecancer or tumor. In some embodiments, the cancer or tumor is ahematologic cancer. In some embodiments, the hematological cancer is a Bcell malignancy. In certain embodiments, the B cell malignancy is B-cellAcute Lymphocytic Leukemia. In certain embodiments, the B cellmalignancy is Chronic Lymphocytic Leukemia, Small Lymphocytic Lymphoma,Mantle Cell Lymphoma, or Non-Hodgkins Lymphomas (Diffuse Large B-cellLymphoma, Follicular Lymphoma). In some embodiments, the hematologicalcancer is a plasma malignancy. In certain embodiments, the plasmamalignancy is multiple myeloma. In some embodiments of any of thepreceding embodiments, the hematological cancer expresses CD19 and CD38(e.g. cells of the cancer express CD19 and CD38).

n some embodiments, the cancer or tumor is a solid-tissue cancer. Insome embodiments, the cancer comprises breast cancer, prostate cancer,pancreatic cancer, lung cancer, kidney cancer, stomach cancer,esophageal cancer, skin cancer, colorectal cancer, or head and neckcancer. In some embodiments, the breast cancer is triple negative breastcancer, the lung cancer is non-small cell lung cancer, the head and neckcancer is head and neck squamous cell cancer, the kidney cancer is renalcell carcinoma, the brain cancer is glioblastoma multiforme, or the skincancer is melanoma.

Disclosed are methods of reducing immunosuppressing B cells affectinganti-tumor immune responses against a tumor of an individual afflictedwith a tumor or cancer comprising administering to the individualafflicted with the tumor or the cancer the composite binding molecule,thereby reducing immunosuppressing B cells affecting the anti-tumorimmune responses. Further disclosed are methods of reducingimmunosuppressive B cells in, adjacent to, or surrounding a tumor of anindividual afflicted with a tumor or cancer comprising administering tothe individual afflicted with the tumor or the cancer the compositebinding molecule, thereby reducing immunosuppressive B cells in,adjacent to, or surrounding the tumor. In some embodiments, the tumorinfiltrating B cells or the immunosuppressive B cells comprise CD19+,CD38+ B cells.

Further disclosed are methods of preparing a cancer treatment for anindividual comprising admixing the composite binding molecule with apharmaceutically acceptable diluent, carrier, or excipient.

Disclosed are also methods of making the composite binding moleculecomprising incubating a cell comprising an expression vector thatcomprises a nucleic acid sequence encoding the composite bindingmolecule in a cell culture medium under conditions sufficient to allowexpression, assembly and secretion of the composite binding moleculeinto the cell culture medium. In some embodiments, the methods compriseisolating and purifying the molecule from the cell culture medium. Suchisolating and purifying can involve a step comprising contacting thecell culture medium or a cell culture medium that has been subjected toone or more purification steps with a resin or column comprisingProtein, Protein G, Protein L, Protein A/G, or any combination thereof,and optionally washing the resin or column to remove one or morenon-composite binding molecules from the cell culture medium or the cellculture medium that has been subjected to one or more purificationsteps.

Provided herein are composite binding molecules comprising a CD19binding component configured to bind CD19 and a CD38 binding componentconfigured to bind CD38, wherein the CD19 binding component comprises anantibody or antigen binding fragment thereof and the CD38 bindingcomponent comprises an antibody or antigen binding fragment thereof. Insome embodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD19 and/or CD38 binding componentcomprise an immunoglobulin heavy and light chain pair, an scFv, a F(ab),a F(ab′)₂, a single domain antibody, a variable region fragment from animmunoglobulin new antigen receptor (VNAR), or a variable region derivedfrom a heavy chain antibody (VHH). In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe CD19 or CD38 binding component comprises an immunoglobulin heavy andlight chain pair. In some embodiments, provided is a composite bindingmolecule of any of the preceding embodiments, wherein the CD19 and CD38binding component comprise an immunoglobulin heavy and light chain pair.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the CD38 binding component comprisesan immunoglobulin heavy chain and an immunoglobulin light chain, whereinthe immunoglobulin heavy chain comprises an HCDR1 amino acid sequenceset forth in any one of SEQ ID NOs: 71-75, an HCDR2 amino acid sequenceset forth in any one of SEQ ID NOs: 81-85, or 150-155, an HCDR3 aminoacid sequence set forth in any one of SEQ ID NOs: 91-95; and theimmunoglobulin light chain comprises an LCDR1 amino acid sequence setforth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequenceset forth in any one of SEQ ID NOs: 111-115, and/or an LCDR3 amino acidsequence set forth in any one of SEQ ID NOs: 121-125; and wherein theCD19 binding component comprises an immunoglobulin heavy chain and animmunoglobulin light chain, wherein the immunoglobulin heavy chaincomprises an HCDR1 amino acid sequence set forth in any one of SEQ IDNOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ IDNOs: 21-25, an HCDR3 amino acid sequence set forth in any one of SEQ IDNOs: 31-35; and the immunoglobulin light chain comprises an LCDR1 aminoacid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and/oran LCDR3 amino acid sequence set forth in any one of SEQ ID NOs:121-125. In some embodiments, provided is a composite binding moleculeof any of the preceding embodiments, wherein the CD 38 binding componentcomprises an immunoglobulin heavy chain comprising an amino acidsequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:3; and the immunoglobulin light chain having at least about 90%, 95%,97%, 99% identity to SEQ ID NO: 4; and/or wherein the CD19 bindingcomponent comprises an immunoglobulin heavy chain comprising an aminoacid sequence having at least about 90%, 95%, 97%, 99% identity to SEQID NO: 1; and an immunoglobulin light chain having at least about 90%,95%, 97%, 99% identity to SEQ ID NO: 4. In some embodiments, provided isa composite binding molecule of any of the preceding embodiments,wherein the immunoglobulin heavy chain comprises an amino acid sequenceidentical to that set forth in SEQ ID NO: 3 or 5; and the immunoglobulinlight chain comprises an amino acid sequence identical to that set forthin SEQ ID NO: 4; and/or wherein the immunoglobulin heavy chain comprisesan amino acid sequence identical to that set forth in SEQ ID NO: 1 or 6;and the immunoglobulin light chain comprises an amino acid sequenceidentical to that set forth in SEQ ID NO: 4.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the composite binding molecule is acommon light chain bispecific IgG. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe CD 38 binding component comprises an immunoglobulin heavy chaincomprising an HCDR1 amino acid sequence set forth in any one of SEQ IDNOs: 71-75, an HCDR2 amino acid sequence set forth in any one of SEQ IDNOs: 81-85, or 150-155, an HCDR3 amino acid sequence set forth in anyone of SEQ ID NOs: 91-95; and the immunoglobulin light chain comprisesan LCDR1 amino acid sequence set forth in any one of SEQ ID NOs:101-105, an LCDR2 amino acid sequence set forth in any one of SEQ IDNOs: 111-115, and/or an LCDR3 amino acid sequence set forth in any oneof SEQ ID NOs: 121-125; and wherein the CD 19 binding componentcomprises an immunoglobulin heavy chain comprising an HCDR1 amino acidsequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acidsequence set forth in any one of SEQ ID NOs: 21-25, an HCDR3 amino acidsequence set forth in any one of SEQ ID NOs: 31-35; and theimmunoglobulin light chain comprises an LCDR1 amino acid sequence setforth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence setforth in any one of SEQ ID NOs: 51-55, and/or an LCDR3 amino acidsequence set forth in any one of SEQ ID NOs: 61-65. In some embodiments,provided is a composite binding molecule of any of the precedingembodiments, wherein the immunoglobulin heavy chain comprises an aminoacid sequence having at least about 90%, 95%, 97%, 99% identity to SEQID NO: 3 or 5; and the immunoglobulin light chain having at least about90%, 95%, 97%, 99% identity to SEQ ID NO: 4; and/or wherein theimmunoglobulin heavy chain comprises an amino acid sequence having atleast about 90%, 95%, 97%, 99% identity to SEQ ID NO: 1 or 7; and theimmunoglobulin light chain having at least about 90%, 95%, 97%, 99%identity to SEQ ID NO: 2.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the immunoglobulin heavy chaincomprises an amino acid sequence identical to that set forth in SEQ IDNO: 3 or 5; and the immunoglobulin light chain comprises an amino acidsequence identical to that set forth in SEQ ID NO: 4; and wherein theimmunoglobulin heavy chain comprises an amino acid sequence identical tothat set forth in SEQ ID NO: 1 or 7; and the immunoglobulin light chaincomprises an amino acid sequence identical to that set forth in SEQ IDNO: 2. In some embodiments, provided is a composite binding molecule ofany of the preceding embodiments, wherein the CD19 binding component orCD38 binding component comprise an scFv. In some embodiments, providedis a composite binding molecule of any of the preceding embodiments,wherein the CD19 binding component comprises an scFv. In someembodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD38 binding component comprises anscFv. In some embodiments, provided is a composite binding molecule ofany of the preceding embodiments, wherein the CD19 binding component orCD38 binding component comprise an immunoglobulin heavy-chain/lightchain pair. In some embodiments, provided is a composite bindingmolecule of any of the preceding embodiments, wherein the CD19 bindingcomponent comprises an immunoglobulin heavy-chain/light chain pair. Insome embodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD38 binding component comprises animmunoglobulin heavy-chain/light chain pair.

Further provided are composite binding molecules, wherein the compositebinding molecule comprises a CD38 antigen binding component that bindsCD38 comprising an anti-CD38 immunoglobulin heavy chain variable regionpaired with an anti-CD38 immunoglobulin light chain variable region anda CD19 antigen binding component that binds CD19 comprising an anti-CD19immunoglobulin heavy chain variable region paired with an anti-CD38immunoglobulin light chain variable region, wherein the CD38 antigenbinding component comprises: a) a heavy chain complementaritydetermining region 1 (HCDR1) comprising an amino acid sequence set forthin any one of SEQ ID NOs: 71-75; b) a heavy chain complementaritydetermining region 2 (HCDR2) comprising an amino acid sequence set forthin any one of SEQ ID NOs: 81-85, or 150-155, c) a heavy chaincomplementarity determining region 3 (HCDR3) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 91-95; d) a light chaincomplementarity determining region 1 (LCDR1) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 101-105; e) a light chaincomplementarity determining region 2 (LCDR2) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 111-115; and/or f) a lightchain complementarity determining region 3 (LCDR3) comprising an aminoacid sequence set forth in any one of SEQ ID NOs: 121-125.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the CD19 antigen binding componentcomprises: g) a heavy chain complementarity determining region 1 (HCDR1)comprising an amino acid sequence set forth in any one of SEQ ID NOs:11-15, h) a heavy chain complementarity determining region 2 (HCDR2)comprising an amino acid sequence set forth in any one of SEQ ID NOs:21-25, i) a heavy chain complementarity determining region 3 (HCDR3)comprising an amino acid sequence set forth in any one of SEQ ID NOs:31-35; j) a light chain complementarity determining region 1 (LCDR1)comprising an amino acid sequence set forth in any one of SEQ ID NOs:101-105; k) a light chain complementarity determining region 2 (LCDR2)comprising an amino acid sequence set forth in any one of SEQ ID NOs:111-115; and/or 1) a light chain complementarity determining region 3(LCDR3) comprising an amino acid sequence set forth in any one of SEQ IDNOs: 121-125.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the CD38 antigen binding componentcomprises an immunoglobulin heavy chain variable region comprising anamino acid sequence having at least about 90%, 95%, 97%, 99% identity toSEQ ID NO: 3 or 5; and an immunoglobulin light chain variable regioncomprising an amino acid sequence having at least about 90%, 95%, 97%,99% identity to SEQ ID NO: 4. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe CD38 antigen binding component comprises an immunoglobulin heavychain variable region comprising an amino acid sequence identical to SEQID NO: 3 or 5; and an immunoglobulin light chain variable regioncomprises an amino acid sequence identical to SEQ ID NO: 4. In someembodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD19 antigen binding componentcomprises an anti-CD19 immunoglobulin heavy chain variable regioncomprising an amino acid sequence having at least about 90%, 95%, 97%,99% identity to SEQ ID NO: 1 or 6; and an immunoglobulin light chainvariable region comprising an amino acid sequence having at least about90%, 95%, 97%, 99% identity to SEQ ID NO: 4.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the anti-CD19 antigen bindingcomponent comprises an immunoglobulin heavy chain variable regioncomprising an amino acid sequence identical to SEQ ID NO: 1 or 6; and animmunoglobulin light chain variable region comprises an amino acidsequence identical to SEQ ID NO: 4. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe anti-CD38 immunoglobulin heavy chain variable region furthercomprises a first immunoglobulin heavy chain constant region. In someembodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the anti-CD38 immunoglobulin light chainvariable region further comprises an immunoglobulin light chain constantregion. In some embodiments, provided is a composite binding molecule ofany of the preceding embodiments, wherein the anti-CD19 immunoglobulinheavy chain variable region further comprises a second immunoglobulinheavy chain constant region. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe first immunoglobulin heavy chain constant region and/or the secondimmunoglobulin heavy chain constant region comprises one or more aminoacid substitutions that disfavors homodimerization of the anti-CD38immunoglobulin heavy chain constant region and/or promotesheterodimerization of the first heavy chain constant region and thesecond heavy chain constant region. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe one of the first or second immunoglobulin heavy chain constantregions comprises a T366W substitution (EU numbering), and the other ofthe first or second immunoglobulin heavy chain constant regionscomprises a T366S/L368A/Y407V substitution (EU numbering), such that theheterodimerization of the first and second immunoglobulin heavy chainconstant regions is favored compared to homodimerization of the first orsecond immunoglobulin heavy chain constant regions. In some embodiments,provided is a composite binding molecule of any of the precedingembodiments, wherein a single bispecific binding molecule is formed fromthe CD38 antigen binding component and the CD19 antigen bindingcomponent.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the structure of a common light chain bispecific IgG.

FIG. 2 illustrates the structure of a Fab-Fc:scFv-Fc bispecific IgG.

FIG. 3 illustrates the structure of a Fab-Fc-Fab:Fc bispecific IgG.

FIG. 4 illustrates the structure of a Fab-Fc-scFv:Fab-Fc-scFv bispecificIgG.

FIG. 5 illustrates the structure of a Fab-Fc-scFv:Fc bispecific IgG.

FIG. 6 illustrates the structure of a Fab-Fc-Fab:Fab-Fc bispecific IgG.

FIG. 7 illustrates the structure of an scFv-Fab-Fc:scFv-Fab-Fcbispecific IgG.

FIG. 8 illustrates the structure of a Fab-Fab-Fc:Fab-Fab-Fc bispecificIgG.

FIG. 9 illustrates the structure of a Fab-Fc-Fab:Fab-Fc-Fab bispecificIgG.

FIG. 10 illustrates the structure of a Fab-Fc-scFv:Fab-Fc bispecificIgG.

FIG. 11 illustrates the structure of an scFv-Fab-Fc:Fc Bispecific IgG

FIG. 12A to 12E show binding data of CD19 and CD38 antibodies. FIG. 12Ashows cell surface expression of CD19 and CD38. FIG. 12B and 12C showbinding profiles of CD19 and CD38 antibodies. FIG. 12D and 12E showsbinding of CD19 and CD38 controls. FIG. 12F shows binding profiles ofcells that do not express CD19 and CD38.

FIG. 13A to 13B shows binding data of antibodies to Daudi cells.

FIG. 14A to 14B shows binding data of antibodies to REH cells.

FIG. 15A to 15B shows binding data of antibodies to CD19 transfectedHEK293 cells.

FIG. 16A to 16B shows binding data of antibodies to CD38 transfectedHEK293 cells.

FIG. 17A to 17B shows binding data of antibodies to non-transfected CHOcells.

FIG. 18A to 18B shows data for direct apoptosis on Daudi cells forantibody test articles.

FIG. 19A to 19B shows data for cross-linking induced apoptosis on Daudicells for antibody test articles.

FIG. 20A to 20C shows ADCC data for three donors across antibody testarticles.

FIG. 21A to 21C shows ADCC data for three donors across antibody testarticles.

FIG. 22A to 22B shows CDC profiles across test articles.

FIG. 23 shows ADCP data across antibody test articles.

FIG. 24 shows RBC binding data across antibody test articles.

FIG. 25A to 25B shows hemagglutination profiles for antibody testarticles.

FIG. 26 shows hemolysis data across antibody test articles.

DETAILED DESCRIPTION

Immunosuppressive B-cell populations that suppress the anti-tumor immuneresponse can be generally defined by the presence of more than one cellsurface biomarker. Therapeutics that effectively and specifically targetimmunosuppressive B cells can therefore be used to preventimmunosuppression and/or remove immunosuppression in, adjacent to, orsurrounding a tumor or within a tumor environment. Provided herein arecomposite binding molecules that target immunosuppressive B cells.Furthermore, provided are composite binding molecules comprising a firstbinding component configured to bind a first target and a second bindingcomponent configured to bind a second target, wherein the first targetcomprises a B-cell lineage surface marker, and wherein the second targetcomprises a suppressive B-cell surface marker. Disclosed herein aremultivalent antibodies that specifically bind to B-cell populationsassociated with negative modulation or immunosuppression of ananti-tumor response. Immunosuppressive B cells can comprise or bedefined by cell surface biomarkers CD19 and CD38. The bispecificantibodies provided herein can target both CD19 and CD38 to inhibit thefunction of immune suppressive B cells. In certain instances, thefunction of immunosuppressive B cells comprises the release of IL10, IL35, TGF-beta, or a combination thereof. Multivalent or bispecificantibodies targeting CD19 and CD38 can also be used for treatingtumorigenic conditions and/or cancers associated with immunosuppressiveB cells and/or immune dysfunction.

The term “immunosuppression” or “immunodepression” or “negative immunemodulation”, as used herein, refers to the reduction or suppression ofthe immune system function, i.e. immunosuppression generally denotes astate when immune system function is reduced or absent. In certaininstances, immunosuppression generally denotes a state when immunesystem function against a tumor or within, surrounding, or adjacent tothe tumor microenvironment is reduced or absent. The whole immuneresponse may be depressed, the immune response within a local orspecific region may be reduced, or a particular population ofimmunologically active lymphocytes may be selectively affected.Antigen-specific immunosuppression may be the result of deletion orsuppression of a particular population of antigen-specific cells, or theresult of enhanced regulation of the immune response by antigen-specificsuppressor cells. References to immunosuppressive B cells refer to Bcells or B-cell populations that exert negative modulation on the immuneresponse and can be identified by specific surface markers associatedwith such populations, such as CD38. In certain instances,immunosuppression can be identified by the presence or release of IL-10,IL-35, TGF-beta, or a combination thereof. In certain instances,immunosuppression can be identified by the presence or release by Bcells of IL-10, IL-35, TGF-beta, or a combination thereof.

As used herein, the term “cancer” can refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Cancer can also include, but is not limited to,hematological tumors and/or solid tumors. Cancer can refer to diseasesof the blood, bones, organs, skin tissues and vascular system, includingbut not limited to bladder, blood, bones, brain, breast, cervix, chest,colon, endometrium, esophagus, eyes, head, kidneys, liver, lungs, lymphnodes, mouth, neck, ovaries, pancreas, prostate, rectum, kidney, skin,stomach, testes, throat and uterus. Specific cancers include, but arenot limited to, leukemia (acute lymphocytic leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronicbone marrow Chronic myelogenous leukemia (CML), hairy cell leukemia,mature B-cell tumor (small lymphocytic lymphoma, B-cell pro-lymphocyticleukemia, lymphoplasmacytic lymphoma (such as Waldenstrom's giant ball)Proteinemia or indolent lymphoma), spleen marginal zone lymphoma, plasmacell myeloma, plasma cell leukemia, plasmacytoma, peri-implantimmunoglobulin deposition, heavy chain disease, extranodal marginal zoneB-cell lymphoma MALT lymphoma), nodal marginal zone B cell lymphoma(NMZL), gastrointestinal tumor (e.g., gastrointestinal stromal tumor(GIST)), follicular lymphoma, mantle cell lymphoma/leukemia, DiffuseB-cell lymphoma, mediastinal (thymus) large B-cell lymphoma,intravascular large B-cell lymphoma, primary exudative lymphoma, andBurkitt's lymphoma (Burkitt lymphoma), mature T cells and natural killercell (NK) tumors (pre-lymphocytic leukemia, T-cell large lymphocyticleukemia, invasive NK cell leukemia, adult T-cell leukemia/lymphoma,Extranodal NK/T-cell lymphoma, enteropathic T-cell lymphoma,hepatosplenic T-cell lymphoma, blastic NK cell lymphoma, mycosisfungoides (Sezary syndrome), primary Skin degenerative large celllymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma,unspecified peripheral T-cell lymphoma and degenerative large celllymphoma, Hodgkin's lymphoma (nodular sclerosis, mixed cell type,lymphocyte rich type, lymphocyte depleted or unreduced type, nodularlymphocyte type), myeloma (multiple myeloma, inert myeloma, smolderingmyeloma)), chronic myeloproliferative diseases,myelodysplasia/myeloproliferative diseases, myelodysplastic syndromes,lymphoproliferative disorders associated with immunodeficiency,histiocytic and dendritic cell tumors, Hypercytosis, chondrosarcoma,Ewing sarcoma, fibrosarcoma, malignant giant cell tumor, myeloma bonedisease, osteosarcoma, breast cancer (hormone dependent, non-hormonedependent), gynecological cancer (child Cervical, endometrial, fallopiantube, gestational trophoblastic disease, ovary, peritoneum, uterus,vagina and vulva), basal cell carcinoma (BCC), squamous cell carcinoma(SCC), malignant melanoma, protuberous cutaneous fibrosarcoma, Merkelcell carcinoma, Kaposi's sarcoma, astrocytoma, hair cell astrocytoma,embryonic hair growth neuroepithelial neoplasia, oligodendroglioma,Ependymoma, glioblastoma multiforme, mixed glioma, oligodendrocyteastrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, embryonaltissue tumor, teratoma, Malignant mesothelioma (peritoneal mesothelioma,pericardial mesothelioma, pleural mesothelioma),gastric-intestinal-pancreatic or gastrointestinal pancreaticneuroendocrine tumor (GEP-NET), carcinoid tumor, pancreatic endocrinetumor (PET)), colorectal adenocarcinoma, knot Rectal cancer, invasiveneuroendocrine tumor, leiomyosarcoma, mucinous adenocarcinoma, signetring cell adenocarcinoma, hepatocellular carcinoma, hepatobiliary livercancer, hepatic blastoma, hemangioma, hepatic adenoma, focal nodularhyperplasia (nodular regenerative hyperplasia, hamartoma), non-smallcell lung cancer (NSCLC) (squamous cell lung cancer, adenocarcinoma,large cell lung cancer), small cell lung cancer, thyroid cancer,prostate cancer (hormone refractory, non-androgen dependent Sex,androgen-dependent, hormone-insensitive), renal cell carcinoma and softtissue sarcoma (fibrosarcoma, malignant fibrous histiocytoma, cutaneousfibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma,angiosarcoma, synovial sarcoma, malignant Peripheral nerve sheathtumor/neurofibrosarcoma, extra-osseous osteosarcoma).

The term “CD19” or “Cluster of Differentiation 19” (also known as B4,T-cell surface antigen Leu-12, and CVID3) refers to a B-cell lineagesurface biomarker or transmembrane protein that in humans is encoded bythe gene CD19. CD19 can function as coreceptor for the B-cell antigenreceptor complex (BCR) on B-lymphocytes, which decreases the thresholdfor activation of downstream signaling pathways and for triggering Bcell responses to antigens. Structurally, a CD19 amino acid sequence hasat least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity with the amino acid sequence, e.g., of GenBankaccession no. NM_001178098.2→NP_001171569.1 or NM_001770.6→NP_001761.3over a sequence length of at least 50, 100, 150, 200, 250, 300, 350,400, 450, 500 amino acids or over the full length of the polypeptide.Structurally, a CD19 nucleic acid sequence has at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity withthe nucleic acid sequence, e.g., of GenBank accession no. NG_007275.1 orNCBI Gene ID 930, over a sequence length of at least 300, 500, 750,1000, 1250, 1500 nucleic acids or over the full length of thepolynucleotide. The sequence alignments can be performed using anyalignment algorithm known in the art, e.g., BLAST, ALIGN, set to defaultsettings.

The term “CD38” or “Cluster of Differentiation 38” (also known asADPRC1) refers to a B-cell surface biomarker or transmembrane proteinthat in humans is encoded by the gene CD38. CD38 can function in B-cellsignaling that leads to cellular activation and proliferation.Structurally, a CD38 amino acid sequence has at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity withthe amino acid sequence, e.g., of GenBank accession no.NM_001775.4→NP_001766.2 over a sequence length of at least 50, 100, 150,200, 250, amino acids or over the full length of the polypeptide.Structurally, an CD19 nucleic acid sequence has at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity withthe nucleic acid sequence, e.g., of GenBank accession no. NC_000004.12or NCBI Gene ID 952, over a sequence length of at least 300, 500, 750nucleic acids or over the full length of the polynucleotide. Thesequence alignments can be performed using any alignment algorithm knownin the art, e.g., BLAST, ALIGN, set to default settings.

The term “antibody” herein is used in the broadest sense and includesmultivalent or bispecific antibodies and monoclonal antibodies,including intact antibodies and functional (antigen-binding) antibodyfragments thereof, including fragment antigen binding (Fab) fragments,F(ab′)2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG)fragments, single chain antibody fragments, including single chainvariable fragments (sFv or scFv), and single domain antibodies (e.g.,sdAb, sdFv, nanobody) fragments. The term encompasses geneticallyengineered and/or otherwise modified forms of immunoglobulins, such asintrabodies, peptibodies, chimeric antibodies, fully human antibodies,humanized antibodies, and heteroconjugate antibodies, multispecific,e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies,tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term“antibody” should be understood to encompass functional antibodyfragments thereof. The term also encompasses intact or full- lengthantibodies, including antibodies of any class or sub-class, includingIgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The antibody cancomprise a human IgG1 constant region. The antibody can comprise a humanIgG4 constant region.

Among the provided antibodies are multispecific or multivalentantibodies (for example, bispecific antibodies and polyreactiveantibodies) and antibody fragments thereof. The antibodies includeantibody-conjugates and molecules comprising the antibodies, such aschimeric molecules. Thus, an antibody includes, but is not limited to,full-length and native antibodies, as well as fragments and portionthereof retaining the binding specificities thereof, such as anyspecific binding portion thereof including those having any number of,immunoglobulin classes and/or isotypes (e.g., IgG1, IgG2, IgG3, IgG4,IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen-binding)fragments or specific binding portions thereof, including but notlimited to Fab, F(ab′)₂, Fv, and scFv (single chain or related entity).A monoclonal antibody is generally one within a composition ofsubstantially homogeneous antibodies; thus, any individual antibodiescomprised within the monoclonal antibody composition are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. A monoclonal antibody can comprise a human IgG1 constantregion or a human IgG4 constant region.

The terms “complementarity determining region,” and “CDR,” which aresynonymous with “hypervariable region” or “HVR,” are known in the artand refer to non-contiguous sequences of amino acids within antibodyvariable regions, which confer antigen specificity and/or bindingaffinity. In general, there are three CDRs in each heavy chain variableregion (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chainvariable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR”are known in the art to refer to the non-CDR portions of the variableregions of the heavy and light chains. In general, there are four FRs ineach full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, andFR-H4), and four FRs in each full-length light chain variable region(FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequenceboundaries of a given CDR or FR can be readily determined using any of anumber of well-known schemes, including those described by Kabat et al.(1991), “Sequences of Proteins of Immunological Interest,” 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD(“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948(“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis andbinding site topography,” J Mol. Biol. 262, 732-745.” (“Contact”numbering scheme); Lefranc MP et al., “IMGT unique numbering forimmunoglobulin and T cell receptor variable domains and Ig superfamilyV-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT”numbering scheme); Honegger A and Pluckthun A, “Yet another numberingscheme for immunoglobulin variable domains: an automatic modeling andanalysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numberingscheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm formodelling antibodies on the WEB,” Protein Eng. 2000 Dec;13(12):819-24(“AbM” numbering scheme. In certain embodiments, the CDRs of theantibodies described herein can be defined by a method selected fromKabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

The boundaries of a given CDR or FR may vary depending on the schemeused for identification. For example, the Kabat scheme is based onstructural alignments, while the Chothia scheme is based on structuralinformation. Numbering for both the Kabat and Chothia schemes is basedupon the most common antibody region sequence lengths, with insertionsaccommodated by insertion letters, for example, “30a,” and deletionsappearing in some antibodies. The two schemes place certain insertionsand deletions (“indels”) at different positions, resulting indifferential numbering. The Contact scheme is based on analysis ofcomplex crystal structures and is similar in many respects to theChothia numbering scheme.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (V_(H) and V_(L), respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three CDRs (See e.g., Kindt et al. KubyImmunology, 6th ed., W. H. Freeman and Co., page 91(2007)). A singleV_(H) or V_(L) domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a V_(H) or V_(L) domain from an antibody that bindsthe antigen to screen a library of complementary V_(L) or V_(H) domains,respectively (See e.g., Portolano et al., J. Immunol. 150:880-887(1993); Clarkson et al., Nature 352:624-628 (1991)).

Among the provided antibodies are antibody fragments. An “antibodyfragment” can refer to a molecule other than an intact antibody thatcomprises a portion of an intact antibody that binds the antigen towhich the intact antibody binds. Examples of antibody fragments include,but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies;linear antibodies; single-chain antibody molecules (e.g. scFv or sFv);and multispecific antibodies formed from antibody fragments. Inparticular embodiments, the antibodies are single-chain antibodyfragments comprising a variable heavy chain region and/or a variablelight chain region, such as scFvs. Antibody fragments can be made byvarious techniques, including but not limited to proteolytic digestionof an intact antibody as well as production by recombinant host cells.In some embodiments, the antibodies are recombinantly-producedfragments, such as fragments comprising arrangements that do not occurnaturally, such as those with two or more antibody regions or chainsjoined by synthetic linkers, e.g., polypeptide linkers, and/or thosethat are not produced by enzyme digestion of a naturally-occurringintact antibody.

Herein a molecule, peptide, polypeptide, antibody, or antibody fragmentcan be referred to as “bispecific” or “dual-specific” includinggrammatical equivalents. A bispecific molecule possesses the ability tospecifically bind to at least two structurally distinct targets. Thespecific binding may be the result of two distinct binding moieties thatare structurally distinct at the molecular level, including but notlimited to distinct non-identical amino acid sequences; or a singlebinding moiety that is able to specifically bind to two structurallydistinct targets with high affinity (e.g., with a KD less than about1×10⁻⁶). A molecule, peptide, polypeptide, antibody, or antibodyfragment referred to as “multi-specific” refers to a molecule thatpossesses the ability to specifically bind to at least threestructurally distinct targets. A “bispecific antibody” includinggrammatical equivalents refers to a bispecific molecule that preservesat least one fragment of an antibody able to specifically bind a target,for example, a variable region, heavy or light chain, or one or morecomplementarity determining regions from an antibody molecule. A“multi-specific antibody” including grammatical equivalents refers to amulti-specific molecule that preserves at least one fragment of anantibody able to specifically bind with a target, for example, avariable region, heavy or light chain, or complementarity determiningregion from an antibody molecule.

A “linker” herein is also referred to as “linker sequence” “spacer”“tethering sequence” or grammatical equivalents thereof. A “linker” asreferred herein connects two distinct molecules that by themselvespossess target binding, catalytic activity, or are naturally expressedand assembled as separate polypeptides. For example, two distinctbinding moieties or a heavy-chain/light-chain pair. A number ofstrategies may be used to covalently link molecules together. Theseinclude but are not limited to polypeptide linkages between N- andC-termini of proteins or protein domains, linkage via disulfide bonds,and linkage via chemical cross-linking reagents. In one aspect of thisembodiment, the linker is a peptide bond, generated by recombinanttechniques or peptide synthesis. The linker peptide may predominantlyinclude the following amino acid residues: Gly, Ser, Ala, or Thr. Thelinker peptide should have a length that is adequate to link twomolecules in such a way that they assume the correct conformationrelative to one another so that they retain the desired activity. In oneembodiment, the linker is from about 1 to 50 amino acids in length orabout 1 to 30 amino acids in length. In one embodiment, linkers of 1 to20 amino acids in length may be used. Useful linkers includeglycine-serine polymers, including for example (GS)n, (GSGGS)n (SEQ IDNO: 224), (GGGGS)n (SEQ ID NO: 225), and (GGGS)n (SEQ ID NO: 226), wheren is an integer of at least one, glycine-alanine polymers,alanine-serine polymers, and other flexible linkers. Exemplary, linkersfor linking antibody fragments or single chain variable fragments caninclude AAEPKSS (SEQ ID NO: 227), AAEPKSSDKTHTCPPCP (SEQ ID NO: 228),GGGG (SEQ ID NO: 229), or GGGGDKTHTCPPCP (SEQ ID NO: 230).Alternatively, a variety of non-proteinaceous polymers, including butnot limited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers, that is may find use as linkers.

“Fragment-based” bispecific antibodies or bispecific antibodiescomprising a “single chain variable fragment” or “scFv” of thisdisclosure can refer to a single chain antibody, or fragment thereof,that comprises two binding moieties and a linker connecting the twobinding moieties. The linker may be a polypeptide linker or other linkerof suitable flexibility so as not to inhibit binding of either targetingmoiety. Fragment based bispecific antibody formats include tandem VHHantibodies, tandem scFvs, scFv-Fabs, F(ab)₂, dual-affinity retargetingantibodies (DARTs). Such fragment-based antibodies can be furthermanipulated to comprise additional binding moieties with specificity fora given target e.g., A₂: B₁, A₁:B₂ or A2: B₂, or with fragments of an Fcregion to improve pharmacokinetics or promote ADCC, ADCP, or CDC.

A “binding moiety” refers to a portion of a molecule, peptide,polypeptide, antibody, or antibody fragment that mediates specificbinding to a recited target or antigen or epitope. By way of example,the binding moiety of an antibody may comprise a heavy-chain/light-chainvariable region pair or one or more complementarity determining regions(CDRs).

A “target” as referred to herein refers to the portion of a moleculethat participates with a binding moiety of a molecule, peptide,polypeptide, antibody, or antibody fragment. A target can comprise anamino acid sequence and/or a carbohydrate, lipid or other chemicalentity. An “antigen” is a target comprising a portion that is able to bebound by an adaptive immune molecule such as an antibody or antibodyfragment, B-cell receptor, or T-cell receptor.

The “valency” of a bispecific or multi-specific molecule refers to thenumber of targets a recited molecule, peptide, polypeptide, antibody, orantibody fragment is able to bind. For instance, a molecule that ismonovalent is able to bind to one molecule of a specific target, abivalent molecule is able to bind to two molecules, and a tetravalentmolecule is able to bind four targets. A bispecific, bivalent molecule,for example, is one that can bind to two targets and to two structurallydifferent targets. For example, a bispecific, bivalent molecule whenplaced into contact with a solution comprising target A and target B maybind A₂, B₂ or A:B.

A “humanized” antibody is an antibody in which all or substantially allCDR amino acid residues are derived from non-human CDRs and all orsubstantially all FR amino acid residues are derived from human FRs. Ahumanized antibody optionally can include at least a portion of anantibody constant region derived from a human antibody. A “humanizedform” of a non-human antibody refers to a variant of the non-humanantibody that has undergone humanization, typically to reduceimmunogenicity to humans, while retaining the specificity and affinityof the parental non-human antibody. In some embodiments, some FRresidues in a humanized antibody are substituted with correspondingresidues from a non-human antibody (e.g., the antibody from which theCDR residues are derived), e.g., to restore or improve antibodyspecificity or affinity.

Among the provided antibodies are human antibodies. A “human antibody”is an antibody with an amino acid sequence corresponding to that of anantibody produced by a human or a human cell, or non-human source thatutilizes human antibody repertoires or other human antibody-encodingsequences, including human antibody libraries. The term excludeshumanized forms of non-human antibodies comprising non-humanantigen-binding regions, such as those in which all or substantially allCDRs are non-human. Human antibodies may be prepared by administering animmunogen to a transgenic animal that has been modified to produceintact human antibodies or intact antibodies with human variable regionsin response to antigenic challenge. Such animals typically contain allor a portion of the human immunoglobulin loci, which replace theendogenous immunoglobulin loci, or which are present extrachromosomallyor integrated randomly into the animal's chromosomes. In such transgenicanimals, the endogenous immunoglobulin loci have generally beeninactivated. Human antibodies also may be derived from human antibodylibraries, including phage display and cell-free libraries, containingantibody-encoding sequences derived from a human repertoire.

“ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein, refers to the cell-mediated reaction wherein nonspecificcytotoxic cells that express FcγRs recognize bound antibody on a targetcell and subsequently cause lysis of the target cell. ADCC can becorrelated with binding to FcγRIIIa wherein increased binding toFcγRIIIa leads to an increase in ADCC activity. “ADCP” or antibodydependent cell-mediated phagocytosis, as used herein, can refer to thecell-mediated reaction wherein nonspecific cytotoxic cells that expressFcγRs recognize bound antibody on a target cell and subsequently causephagocytosis of the target cell.

The terms “polypeptide” and “protein” are used interchangeably andrefers to a polymer of amino acid residues, and are not limited to aminimum length. Polypeptides, including the provided antibodies andantibody chains and other peptides, e.g., linkers and binding peptides,can include amino acid residues including natural and/or non-naturalamino acid residues. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. In someaspects, the polypeptides can contain modifications with respect to anative or natural sequence, as long as the protein maintains the desiredactivity. These modifications can be deliberate, as throughsite-directed mutagenesis, or can be accidental, such as throughmutations of hosts which produce the proteins or errors due to PCRamplification.

Percent (%) sequence identity with respect to a reference polypeptidesequence is the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in thereference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that areknown for instance, using publicly available computer software such asBLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriateparameters for aligning sequences are able to be determined, includingalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc., and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available from Genentech,Inc., South San Francisco, Calif., or may be compiled from the sourcecode. The ALIGN-2 program should be compiled for use on a UNIX operatingsystem, including digital UNIX V4.0D. All sequence comparison parametersare set by the ALIGN-2 program and do not vary. In situations whereALIGN-2 is employed for amino acid sequence comparisons, the % aminoacid sequence identity of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows: 100 times the fraction X/Y, whereX is the number of amino acid residues scored as identical matches bythe sequence alignment program ALIGN-2 in that program's alignment of Aand B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

Amino acid sequence variants of the antibodies provided herein can becontemplated and conceived. A variant typically differs from apolypeptide specifically disclosed herein in one or more substitutions,deletions, additions and/or insertions. Such variants can be naturallyoccurring or can be synthetically generated, for example, by modifyingone or more of the above polypeptide sequences of the invention andevaluating one or more biological activities of the polypeptide asdescribed herein and/or using any of a number of known techniques. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibody amino acid sequence variantsof an antibody can be prepared by introducing appropriate modificationsinto the nucleotide sequence encoding the antibody, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of residues within the aminoacid sequences of the antibody. Any combination of deletion, insertion,and substitution can be made to arrive at the final construct, providedthat the final construct possesses the desired characteristics, e.g.,antigen-binding. Antibody variants having one or more amino acidsubstitutions can be provided. Sites of interest for mutagenesis bysubstitution include the CDRs and FRs. Amino acid substitutions can beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

This disclosure also provides for “immunoconjugates” or “antibodyconjugates” or “antibody-drug conjugates” that refer to an antibodyconjugated to one or more heterologous molecule(s). For example, animmunoconjugate can comprise an antibody conjugated to one or morecytotoxic agents, such as chemotherapeutic agents or drugs, growthinhibitory agents, protein domains, toxins (e.g., protein toxins,enzymatically active toxins of bacterial, fungal, plant, or animalorigin, or fragments thereof), or radioactive isotopes. In someembodiments, an immunoconjugate can comprise the composite bindingmolecule disclosed herein, or fragment thereof (e.g., an scFv).

The antibodies described herein can be encoded by a nucleic acid. Anucleic acid is a type of polynucleotide comprising two or morenucleotide bases. In certain embodiments, the nucleic acid is acomponent of a vector that can be used to transfer the polypeptideencoding polynucleotide into a cell. As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. One type of vector is agenomic integrated vector, or “integrated vector,” which can becomeintegrated into the chromosomal DNA of the host cell. Another type ofvector is an “episomal” vector, e.g., a nucleic acid capable ofextra-chromosomal replication. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors.” Suitable vectors comprise plasmids,bacterial artificial chromosomes, yeast artificial chromosomes, viralvectors and the like. In the expression vectors regulatory elements suchas promoters, enhancers, polyadenylation signals for use in controllingtranscription can be derived from mammalian, microbial, viral or insectgenes. The ability to replicate in a host, usually conferred by anorigin of replication, and a selection gene to facilitate recognition oftransformants may additionally be incorporated. Vectors derived fromviruses, such as lentiviruses, retroviruses, adenoviruses,adeno-associated viruses, and the like, may be employed. Plasmid vectorscan be linearized for integration into a chromosomal location. Vectorscan comprise sequences that direct site-specific integration into adefined location or restricted set of sites in the genome (e.g.,AttP-AttB recombination). Additionally, vectors can comprise sequencesderived from transposable elements.

As used herein, the terms “homologous,” “homology,” or “percenthomology” when used herein to describe to an amino acid sequence or anucleic acid sequence, relative to a reference sequence, can bedetermined using the formula described by Karlin and Altschul (Proc.Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl.Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated intothe basic local alignment search tool (BLAST) programs of Altschul etal. (J. Mol. Biol. 215: 403-410, 1990). Percent homology of sequencescan be determined using the most recent version of BLAST, as of thefiling date of this application.

The nucleic acids encoding the antibodies described herein can be usedto infect, transfect, transform, or otherwise render a suitable celltransgenic for the nucleic acid, thus enabling the production ofantibodies for commercial or therapeutic uses. Standard cell lines andmethods for the production of antibodies from a large-scale cell cultureare known in the art. See e.g., Li et al., “Cell culture processes formonoclonal antibody production.” Mabs. 2010 Sep-Oct; 2(5): 466-477. Incertain embodiments, the cell is a Eukaryotic cell. In certainembodiments, the Eukaryotic cell is a mammalian cell. In certainembodiments, the mammalian cell is a cell line useful for producingantibodies is a Chines Hamster Ovary cell (CHO) cell, an NSO murinemyeloma cell, or a PER.C6® cell. In certain embodiments, the nucleicacid encoding the antibody is integrated into a genomic locus of a celluseful for producing antibodies. In certain embodiments, describedherein is a method of making an antibody comprising culturing a cellcomprising a nucleic acid encoding an antibody under conditions in vitrosufficient to allow production and secretion of said antibody.

As used herein the term “individual,” “patient,” or “subject” refers toindividuals diagnosed with, suspected of being afflicted with, orat-risk of developing at least one disease for which the describedcompositions and method are useful for treating. In certain embodiments,the individual is a mammal. In certain embodiments, the mammal is amouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama,alpaca, or yak. In certain embodiments, the individual is a human.

As used herein, the term “about” used to modify a specific number refersto that number plus or minus 10% of that number. The term “about”modifying a range refers to that range minus 10% of its lowest value andplus 10% of its greatest value.

As used herein, the terms “treatment” or “treating” are used inreference to a pharmaceutical or other intervention regimen used forobtaining beneficial or desired results in the recipient. Beneficial ordesired results include but are not limited to a therapeutic benefitand/or a prophylactic benefit. A therapeutic benefit may refer toeradication or amelioration of symptoms or of an underlying disorderbeing treated. Also, a therapeutic benefit can be achieved with theeradication or amelioration of one or more of the physiological symptomsassociated with the underlying disorder such that an improvement isobserved in the subject, notwithstanding that the subject may still beafflicted with the underlying disorder. A prophylactic effect includesdelaying, preventing, or eliminating the appearance of a disease orcondition, delaying or eliminating the onset of symptoms of a disease orcondition, slowing, halting, or reversing the progression of a diseaseor condition, or any combination thereof. For prophylactic benefit, asubject at risk of developing a particular disease, or to a subjectreporting one or more of the physiological symptoms of a disease mayundergo treatment, even though a diagnosis of this disease may not havebeen made. Skilled artisans will recognize that given a population ofpotential individuals for treatment not all will respond or respondequally to the treatment. Such individuals are considered treated.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Bispecific Molecules

Provided herein are bispecific or multivalent or composite bindingmolecules comprising a first binding component configured to bind afirst target and a second binding component configured to bind a secondtarget, wherein the first target comprises a B-cell lineage surfacemarker, and wherein the second target comprises a suppressive B-cellsurface marker. Immunosuppressive B cells or B-cell populations cancomprise a B-cell linage surface biomarker and a suppressive B-cellsurface biomarker. The B-cell lineage surface markers can comprise CD19,CD138, IgA, or CD45. Immunosuppressive B-cell surface markers cancomprise IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latentTGF-beta (e.g., TGF-beta LAP). In some embodiments, the B-cell lineagesurface marker comprises CD19. In certain embodiments, the B-celllineage surface marker consists of CD19. In some embodiments, thesuppressive B-cell surface marker comprises CD38. In certainembodiments, the suppressive B-cell surface marker consists of CD38. Incertain embodiments, the composite binding molecule binds to CD38 andCD19.

A multivalent or bispecific or composite binding molecule possesses theability to specifically bind to at least two structurally distincttargets. The specific binding may be the result of two distinct bindingmoieties that are structurally distinct at the molecular level,including but not limited to distinct non-identical amino acidsequences; or a single binding moiety that is able to specifically bindto two structurally distinct targets. A molecule, peptide, polypeptide,antibody, or antibody fragment referred to as “multi-specific” or“multivalent” or “bispecific” can refer to a molecule that possesses theability to specifically bind to at least two structurally distincttargets. In some embodiments, the first or the second binding componentof the composite binding molecule comprises a polypeptide. In certainembodiments, the first or the second binding component consists of apolypeptide. In some embodiments, the first and the second bindingcomponent of the composite binding molecule comprises a polypeptide. Incertain embodiments, the first and the second binding component consistof a polypeptide. In certain embodiments, the polypeptide of the firstor second binding component comprises an amino acid sequence at least100 amino acid residues in length. In certain embodiments, thepolypeptide of the first and second binding component comprise an aminoacid sequence at least 100 amino acid residues in length.

A bispecific molecule can be a bispecific antibody that preserves atleast one fragment of an antibody able to specifically bind with atarget, for example, a variable region, heavy or light chain, or one ormore complementarity determining regions from an antibody molecule. Insome embodiments, the composite binding molecule described herein is abispecific antibody and/or dual antigen-binding fragment thereof.Bispecific antibodies possess the ability to bind to two structurallydistinct targets or antigens. In some embodiments, the bispecificantibody comprises a first binding component configured to bind a firsttarget and a second binding component configured to bind a secondtarget, wherein the first target comprises a B-cell lineage surfacemarker (e.g. CD19, CD138, IgA, or CD45), and wherein the second targetcomprises a suppressive B-cell surface marker (e.g. IgD, CD1, CD5, CD21,CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-betaLAP)). In some embodiments, the B-cell lineage surface marker comprisesCD19. In certain embodiments, the B-cell lineage surface marker consistsof CD19. In some embodiments, the suppressive B-cell surface markercomprises CD38. In certain embodiments, the suppressive B-cell surfacemarker consists of CD38.

Immunosuppressive B cells or immunosuppressive B-cell populations cancomprise cell surface biomarkers CD19 and CD38. Further disclosed hereinare bispecific antibodies that target CD19 and CD38. In someembodiments, the CD19 binding component comprises a variable heavy chain(VH) comprising SEQ ID NO: 1. In certain embodiments, the CD19 bindingcomponent comprises a VH CDR1 region comprising any one of SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. Incertain embodiments, the CD19 binding component comprises a VH CDR2region comprising any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, or SEQ ID NO: 25. In certain embodiments, the CD19binding component comprises a VH CDR3 region comprising any one of SEQID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO:35.

In some embodiments, the CD19 binding component comprises a variablelight chain (VL) comprising SEQ ID NO: 2. In certain embodiments, theCD19 binding component comprises a VL CDR1 region comprising any one ofSEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, or SEQ IDNO: 45. In certain embodiments, the CD19 binding component comprises aVL CDR2 region comprising any one of SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 55. In certain embodiments, theCD19 binding component comprises a VL CDR3 region comprising any one ofSEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, or SEQ IDNO: 65.

In some embodiments, the bispecific antibody comprises a first bindingcomponent, wherein the first binding component comprises an HCDR1 aminoacid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 aminoacid sequence set forth in any one of SEQ ID NOs: 21-25, an HCDR3 aminoacid sequence set forth in any one of SEQ ID NOs: 31-35, an LCDR1 aminoacid sequence set forth in any one of SEQ ID NOs: 41-45, an LCDR2 aminoacid sequence set forth in any one of SEQ ID NOs: 51-55, and/or an LCDR3amino acid sequence set forth in any one of SEQ ID NOs: 61-65.

In some embodiments, the bispecific antibody comprises a CD19 bindingcomponent, wherein the CD19 binding component comprises an HCDR1 aminoacid sequence set forth in SEQ ID NO: 11, an HCDR2 amino acid sequenceset forth in SEQ ID NO: 21, an HCDR3 amino acid sequence set forth inSEQ ID NO: 31, an LCDR1 amino acid sequence set forth in SEQ ID NO: 41,an LCDR2 amino acid sequence set forth in SEQ ID NO: 51, and/or an LCDR3amino acid sequence set forth in SEQ ID NO: 61.

In some embodiments, the bispecific antibody comprises a CD19 bindingcomponent, wherein CD19 first binding component comprises an HCDR1 aminoacid sequence set forth in SEQ ID NO: 12, an HCDR2 amino acid sequenceset forth in SEQ ID NO: 22, an HCDR3 amino acid sequence set forth inSEQ ID NO: 32, an LCDR1 amino acid sequence set forth in SEQ ID NO: 42,an LCDR2 amino acid sequence set forth in SEQ ID NO: 52, and/or an LCDR3amino acid sequence set forth in SEQ ID NO: 62.

In some embodiments, the bispecific antibody comprises a CD19 bindingcomponent, wherein the CD19 binding component comprises an HCDR1 aminoacid sequence set forth in SEQ ID NO: 15, an HCDR2 amino acid sequenceset forth in SEQ ID NO: 25, an HCDR3 amino acid sequence set forth inSEQ ID NO: 35, an LCDR1 amino acid sequence set forth in SEQ ID NO: 45,an LCDR2 amino acid sequence set forth in SEQ ID NO: 55, and/or an LCDR3amino acid sequence set forth in SEQ ID NO: 65.

In some embodiments, the CD19 binding comprises a variable heavy chainand light chain or CDRs corresponding to or derived from Inebilizumab,Tafasitamab, Taplitumomab, Obexelimab, Blinatumomab, Coltuximab,Denintuzumab, or Loncastuximab, MOR208, MEDI-551, XmAb 5871, MDX-1342,or AFM11.

In some embodiments, the CD38 binding component comprises a variableheavy chain (VH) comprising SEQ ID NO: 3. In certain embodiments, theCD19 binding component comprises a VH CDR1 region comprising any one ofSEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 75, or SEQ IDNO: 75. In certain embodiments, the CD19 binding component comprises aVH CDR2 region comprising any one of SEQ ID NO: 81, SEQ ID NO: 82, SEQID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 85. In certain embodiments, theCD19 binding component comprises a VH CDR3 region comprising any one ofSEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ IDNO: 95.

In some embodiments, the CD38 binding component comprises a variablelight chain (VL) comprising SEQ ID NO: 4. In certain embodiments, theCD19 binding component comprises a VL CDR1 region comprising any one ofSEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, or SEQID NO: 105. In certain embodiments, the CD19 binding component comprisesa VL CDR2 region comprising any one of SEQ ID NO: 111, SEQ ID NO: 112,SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 115. In certainembodiments, the CD19 binding component comprises a VL CDR3 regioncomprising any one of SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,SEQ ID NO: 124, or SEQ ID NO: 125.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent, wherein the CD38 binding component comprises an HCDR1 aminoacid sequence set forth in SEQ ID NO: 71, an HCDR2 amino acid sequenceset forth in SEQ ID NO: 81, an HCDR3 amino acid sequence set forth inSEQ ID NO: 91, an LCDR1 amino acid sequence set forth in SEQ ID NO: 101,an LCDR2 amino acid sequence set forth in SEQ ID NO: 111, and/or anLCDR3 amino acid sequence set forth in SEQ ID NO: 121.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent, wherein the CD38 binding component comprises an HCDR1 aminoacid sequence set forth in SEQ ID NO: 72, an HCDR2 amino acid sequenceset forth in SEQ ID NO: 82, an HCDR3 amino acid sequence set forth inSEQ ID NO: 92, an LCDR1 amino acid sequence set forth in SEQ ID NO: 102,an LCDR2 amino acid sequence set forth in SEQ ID NO: 112, and/or anLCDR3 amino acid sequence set forth in SEQ ID NO: 122.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent, wherein the CD38 binding component comprises an HCDR1 aminoacid sequence set forth in SEQ ID NO: 75, an HCDR2 amino acid sequenceset forth in SEQ ID NO: 85, an HCDR3 amino acid sequence set forth inSEQ ID NO: 95, an LCDR1 amino acid sequence set forth in SEQ ID NO: 105,an LCDR2 amino acid sequence set forth in SEQ ID NO: 115, and/or anLCDR3 amino acid sequence set forth in SEQ ID NO: 125.

In some embodiments (e.g., any of the preceding embodiments), the CDR-H2of the CD38 binding component comprises the amino acid residuesP(X1)LG(X2)A (SEQ ID NO: 150), wherein X1 and X2 tolerate amino acidsubstitutions while maintaining binding to CD38. In certain embodiments,X1 and X2 are selected from amino acids that reduce the hydrophobicityof the CDRH2 amino acid sequence. In certain embodiments, the aminoacids that reduce the hydrophobicity include H, Q, T, N, S, G, A, R, K,D, or E. In certain embodiments, the X1 is H and X2 is T.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent and a CD19 binding component, wherein the CD38 bindingcomponent comprises a VH amino acid sequence and a VL amino acidsequence and, wherein the VH amino acid sequence comprises an amino acidsequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ IDNO: 3, and the VL comprises an amino acid sequence at least about 90%,95%, 97%, 98%, or 99% identical to SEQ ID NO: 4; and the CD19 bindingcomponent comprises a VH amino acid sequence and a VL amino acidsequence, wherein the VH amino acid sequence comprises an amino acidsequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ IDNO: 1, and the VL comprises an amino acid sequence at least about 90%,95%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent and a CD19 binding component, wherein the CD38 bindingcomponent comprises a VH amino acid sequence and a VL amino acidsequence and, wherein the VH amino acid sequence comprises an amino acidsequence identical to SEQ ID NO: 3, and the VL comprises an amino acidsequence identical to SEQ ID NO: 4; and the CD19 binding componentcomprises a VH amino acid sequence and a VL amino acid sequence, whereinthe VH amino acid sequence comprises an amino acid sequence identical toSEQ ID NO: 1, and the VL comprises an amino acid sequence identical toSEQ ID NO: 2.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent and a CD19 binding component, wherein the CD38 bindingcomponent comprises a VH amino acid sequence and a VL amino acidsequence and, wherein the VH amino acid sequence comprises an amino acidsequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ IDNOs: 3, 215, or 218-223, and the VL comprises an amino acid sequence atleast about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs: 4 or 223; and the CD19 binding component comprises a VH amino acid sequence anda VL amino acid sequence, wherein the VH amino acid sequence comprisesan amino acid sequence at least about 90%, 95%, 97%, 98%, or 99%identical to SEQ ID NOs: 1, 201, or 216-217, and the VL comprises anamino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identicalto SEQ ID NO: 2. In some embodiments, the CD19 binding componentcomprises a VH amino acid sequence comprising a substitution at A84 andA108. In some embodiments, the substitution comprises A84S and A108L.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent and a CD19 binding component, wherein the CD38 bindingcomponent comprises a VH amino acid sequence and a VL amino acidsequence and, wherein the VH amino acid sequence comprises an amino acidsequence identical to SEQ ID NO: 3, 215, or 218-223, and the VLcomprises an amino acid sequence identical to SEQ ID NO: 4 or 223; andthe CD19 binding component comprises a VH amino acid sequence and a VLamino acid sequence, wherein the VH amino acid sequence comprises anamino acid sequence identical to SEQ ID NO: 1, 201, 216-217 and the VLcomprises an amino acid sequence identical to SEQ ID NO: 2. In someembodiments, the CD19 binding component comprises a VH amino acidsequence comprising a substitution at A84 and A108. In some embodiments,the substitution comprises A84S and A108L.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent and a CD19 binding component, wherein the CD38 bindingcomponent comprises an HCDR1 amino acid sequence set forth in SEQ ID NO:71, an HCDR2 amino acid sequence set forth in SEQ ID NO: 81, an HCDR3amino acid sequence set forth in SEQ ID NO: 91, an LCDR1 amino acidsequence set forth in SEQ ID NO: 101, an LCDR2 amino acid sequence setforth in SEQ ID NO: 111, and/or an LCDR3 amino acid sequence set forthin SEQ ID NO: 121; and the CD19 binding component comprises an HCDR1amino acid sequence set forth in SEQ ID NO: 11, an HCDR2 amino acidsequence set forth in SEQ ID NO: 21, an HCDR3 amino acid sequence setforth in SEQ ID NO: 31, an LCDR1 amino acid sequence set forth in SEQ IDNO: 41, an LCDR2 amino acid sequence set forth in SEQ ID NO: 51, and/oran LCDR3 amino acid sequence set forth in SEQ ID NO: 61.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent and a CD19 binding component, wherein the CD38 bindingcomponent comprises an HCDR1 amino acid sequence set forth in SEQ ID NO:72, an HCDR2 amino acid sequence set forth in SEQ ID NO: 82, an HCDR3amino acid sequence set forth in SEQ ID NO: 92, an LCDR1 amino acidsequence set forth in SEQ ID NO: 102, an LCDR2 amino acid sequence setforth in SEQ ID NO: 112, and/or an LCDR3 amino acid sequence set forthin SEQ ID NO: 122; and the CD19 binding component comprises an HCDR1amino acid sequence set forth in SEQ ID NO: 12, an HCDR2 amino acidsequence set forth in SEQ ID NO: 22, an HCDR3 amino acid sequence setforth in SEQ ID NO: 32, an LCDR1 amino acid sequence set forth in SEQ IDNO: 42, an LCDR2 amino acid sequence set forth in SEQ ID NO: 52, and/oran LCDR3 amino acid sequence set forth in SEQ ID NO: 62.

In some embodiments, when the bispecific comprises a Fab or otherstructure requiring a light chain constant region for the bispecificformat, the VL comprises an amino acid sequence at least about 90%, 95%,97%, 98%, 99% or is identical to any one of SEQ ID NOs: 210 and/or 211.In some embodiments, when the bispecific comprises a Fab or otherstructure requiring a light chain constant region for the bispecificformat, the VL comprises an amino acid sequence identical to any one ofSEQ ID NOs: 210 and/or 211.

In some embodiments, the bispecific antibody comprises a CD38 bindingcomponent and a CD19 binding component, wherein the CD38 bindingcomponent comprises an HCDR1 amino acid sequence set forth in SEQ ID NO:75, an HCDR2 amino acid sequence set forth in SEQ ID NO: 85, an HCDR3amino acid sequence set forth in SEQ ID NO: 95, an LCDR1 amino acidsequence set forth in SEQ ID NO: 105, an LCDR2 amino acid sequence setforth in SEQ ID NO: 115, and/or an LCDR3 amino acid sequence set forthin SEQ ID NO: 125; and the CD19 binding component comprises an HCDR1amino acid sequence set forth in SEQ ID NO: 15, an HCDR2 amino acidsequence set forth in SEQ ID NO: 25, an HCDR3 amino acid sequence setforth in SEQ ID NO: 35, an LCDR1 amino acid sequence set forth in SEQ IDNO: 45, an LCDR2 amino acid sequence set forth in SEQ ID NO: 55, and/oran LCDR3 amino acid sequence set forth in SEQ ID NO: 65.

In some embodiments, the CD38 binding comprises a variable heavy chainand light chain or CDRs corresponding to or derived from Daratumumab orIsatuximab.

Substitutions, insertions, or deletions may occur within one or moreCDRs, wherein the substitutions, insertions, or deletions do notsubstantially reduce antibody binding to antigen. For example,conservative substitutions that do not substantially reduce bindingaffinity may be made in CDRs. Such alterations may be outside of CDR“hotspots”. In some embodiments, of the variant V_(H) and V_(L)sequences, each CDR is unaltered. Amino acid sequence insertions anddeletions include amino- and/or carboxyl-terminal fusions ranging inlength from one residue to polypeptides containing a hundred or moreresidues, as well as intrasequence insertions and deletions of single ormultiple amino acid residues. Examples of terminal insertions include anantibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.Examples of intrasequence insertion variants of the antibody moleculesinclude an insertion of 3 amino acids in the light chain. Examples ofterminal deletions include an antibody with a deletion of 7 or lessamino acids at an end of the light chain.

Alterations (e.g., substitutions) may be made in CDRs, e.g., to improveantibody affinity. Such alterations may be made in CDR encoding codonswith a high mutation rate during somatic maturation (See e.g.,Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resultingvariant can be tested for binding affinity. Affinity maturation (e.g.,using error-prone PCR, chain shuffling, randomization of CDRs, oroligonucleotide-directed mutagenesis) can be used to improve antibodyaffinity (See e.g., Hoogenboom et al. in Methods in Molecular Biology178:1-37 (2001)). CDR residues involved in antigen binding may bespecifically identified, e.g., using alanine scanning mutagenesis ormodeling (See e.g., Cunningham and Wells Science, 244:1081-1085 (1989)).CDR-H3 and CDR-L3 in particular are often targeted. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Antibodies can be altered to increase or decrease their glycosylation(e.g., by altering the amino acid sequence such that one or moreglycosylation sites are created or removed). A carbohydrate attached toan Fc region of an antibody may be altered. Native antibodies frommammalian cells typically comprise a branched, biantennaryoligosaccharide attached by an N-linkage to Asn297 of the CH2 domain ofthe Fc region (See e.g., Wright et al. TIBTECH 15:26-32 (1997)). Theoligosaccharide can be various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, fucose attached to aGlcNAc in the stem of the biantennar oligosaccharide structure.Modifications of the oligosaccharide in an antibody can be made, forexample, to create antibody variants with certain improved properties.Antibody glycosylation variants can have improved ADCC and/or CDCfunction. In some embodiments, antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn297 (See e.g., WO08/077546). Asn297 refers to the asparagine residue located at aboutposition 297 in the Fc region (EU numbering of Fc region residues; Seee.g., Edelman et al. Proc Natl Acad Sci USA. 1969 May; 63(1):78-85).However, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such fucosylation variants canhave improved ADCC function (See e.g., Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004)). Cell lines, e.g., knockout cell lines and methods of their usecan be used to produce defucosylated antibodies, e.g., Lec13 CHO cellsdeficient in protein fucosylation and alpha-1,6-fucosyltransferase gene(FUT8) knockout CHO cells (See e.g., Ripka et al. Arch. Biochem.Biophys. 249:533-545 (1986); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688(2006)). Other antibody glycosylation variants are also included (Seee.g., U .S. Pat. No. 6,602,684).

In some embodiments, the composite binding molecule provided herein hasa dissociation constant (K_(D)) of about 10 μM, 1 μM, 100 nM, 50 nM, 40nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01nM, or 0.001 nM or less (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to10⁻¹³ M, e.g., from 10⁻⁹M to 10⁻¹³ M) for the antibody target. Theantibody target can be a CD19 target, a CD38 target, or a targetcomprising both CD19 and CD38. K_(D) can be measured by any suitableassay. In certain embodiments, KD can be measured using surface plasmonresonance assays (e.g., using a BIACORE®-2000 or a BIACOREg-3000 orOctet®).

In some embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. An Fc region herein is a C-terminalregion of an immunoglobulin heavy chain that contains at least a portionof the constant region. An Fc region includes native sequence Fc regionsand variant Fc regions. The Fc region variant may comprise a human Fcregion sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region)comprising an amino acid modification (e.g., a substitution) at one ormore amino acid positions.

In some instances, the Fc region of an immunoglobulin is important formany important antibody functions (e.g. effector functions), such asantigen-dependent cellular cytotoxicity (ADCC), complement dependentcytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis(ADCP), result in killing of target cells, albeit by differentmechanisms. Accordingly, in some embodiments, the antibodies describedherein comprise the variable domains of the invention combined withconstant domains comprising different Fc regions, selected based on thebiological activities of the antibody for the intended use. In certaininstances, Human IgGs, for example, can be classified into foursubclasses, IgG1, IgG2, IgG3, and IgG4, and each these of thesecomprises an Fc region having a unique profile for binding to one ormore of Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA,FcγRIIC (CD32); FcγRIIIA and FcγRIIIB (CD16) and inhibiting receptorFcγRIIB), and for the first component of complement (C1q). Human IgG1and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIA_(H131), andwith lower affinity to FcγRIIA_(R131)FcγRIIIA_(V158); IgG4 binds toFcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA_(V158) ; and theinhibitory receptor FcγRIIB has a lower affinity for IgG1, IgG2 and IgG3than all other Fcγ receptors. Studies have shown that FcγRI does notbind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Id. Ingeneral, with regard to ADCC activity, human IgG1≥IgG3»IgG4≥IgG2.

In certain embodiments, anti-CD19 or anti-CD38 variable regionsdescribed herein are linked to an Fc that binds to one or moreactivating Fc receptors (FcγRI/CD64, FcγRIIa/CD32 or FcγRIIIa/CD16), andthereby stimulate ADCC and, in some instances, cause target depletion.In certain embodiments, anti-CD19 or anti-CD38 variable regionsdescribed herein are linked to a human IgG1 or IgG3 Fc, i.e., theantibodies are of the IgG1 or IgG3 isotype. In some instances,modifications in the Fc region generate an Fc variant with (a) increasedantibody-dependent cell-mediated cytotoxicity ADCC), (b) increasedcomplement mediated cytotoxicity (CDC), (c) increased affinity for C1qand/or (d) increased affinity for a Fc receptor relative to the parentFc. In some embodiments, the Fc region variants comprise at least oneamino acid modification in the Fc region. Combining amino acidmodifications are also useful. For example, the variant Fc region mayinclude two, three, four, five, etc. substitutions therein, e.g. of thespecific Fc region positions identified herein.

In some embodiments, ADCC activity may be increased by modifying the Fcregion. With regard to ADCC activity, in some instances, human IgG1 andIgG3 shows increased ADCC activation when compared to IgG4 and gG2, soan IgG1 or IgG3 constant domain, rather than an IgG2 or IgG4, is chosenfor use in an antibody where ADCC is desired. In some embodiments, IgG3is selected for activation of FcγRIIIA-expressing NK cells, monocytes ofmacrophages. In certain instances, different IgG isotypes also exhibitdifferential CDC activity, wherein IgG3 and IgG1 show greater CDCactivation than compared to IgG2 or IgG4. lternatively, in someembodiments, the Fc region is modified to increase antibody dependentcellular cytotoxicity (ADCC), antibody-dependent cell-mediatedphagocytosis (ADCP), complement mediated cytotoxicity (CDC), affinityfor C1q, and/or to increase the affinity for an Fcγ receptor bymodifying one or more amino acids at the following positions: 234, 235,236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255,256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280,283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303,305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330,331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388,389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439 (Kabatnumbering). Non-limiting examples of in vitro assays to assess ADCCactivity of a molecule of interest is described in U.S. Pat. No.5,500,362 and 5,821,337. Alternatively, non-radioactive assays methodsmay be employed (e.g., ACTI™ and CytoTox 96® non-radioactivecytotoxicity assays). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC), monocytes, macrophages, andNatural Killer (NK) cells.

Antibodies can have increased half-lives and improved binding to theneonatal Fc receptor (FcRn) (See e.g., US 2005/0014934). Such antibodiescan comprise an Fc region with one or more substitutions therein whichimprove binding of the Fc region to FcRn, and include those withsubstitutions at one or more of Fc region residues: 238, 256, 265, 272,286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 413, 424 or 434 according to the EU numbering system (See e.g.,U.S. Pat. No. 7,371,826). Other examples of Fc region variants are alsocontemplated (See e.g., Duncan & Winter, Nature 322:738-40 (1988); U.S.Pat. Nos. 5,648,260 and 5,624,821; and WO94/29351).

In some embodiments, it may be desirable to create cysteine engineeredantibodies, e.g., “thioMAbs,” in which one or more residues of anantibody are substituted with cysteine residues. In some embodiments,the substituted residues occur at accessible sites of the antibody.Reactive thiol groups can be positioned at sites for conjugation toother moieties, such as drug moieties or linker drug moieties, to createan immunoconjugate. In some embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region.

In some embodiments, an antibody provided herein may be further modifiedto contain additional non-proteinaceous moieties that are known andavailable. The moieties suitable for derivatization of the antibodyinclude but are not limited to water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n vinyl pyrrolidone)polyethyleneglycol, polypropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if two or more polymers are attached, they can be the same ordifferent molecules.

Composite binding molecules or bispecific antibodies can differ based onthe binding moieties associated with these molecules, wherein there arealso several different formats that can be deployed and are envisionedherein. Composite binding molecules or bispecific antibodies cancomprise on antibody fragments, substantially intact antibodies, or acombination thereof In some embodiments, the first or second bindingcomponent comprises an immunoglobulin heavy and light chain pair, anscFv, a F(ab), a F(ab′)₂, a single domain antibody, a variable regionfragment from an immunoglobulin new antigen receptor (VNAR), or avariable region derived from a heavy chain antibody (VHH). In certainembodiments, the first and second binding component comprise animmunoglobulin heavy and light chain pair, an scFv, a F(ab), a F(ab′)₂,a single domain antibody, a variable region fragment from animmunoglobulin new antigen receptor (VNAR), or a variable region derivedfrom a heavy chain antibody (VHH). In some embodiments, the first orsecond binding component comprises an immunoglobulin heavy and lightchain pair. In certain embodiments, the first and second bindingcomponent comprise an immunoglobulin heavy and light chain pair. In someembodiments, the first or second binding component comprises an scFv. Incertain embodiments, the first and second binding component comprise anscFv.

Bispecific antibodies according to this disclosure comprise intactantibody molecules or substantially fully intact antibody molecules, andmay be asymmetric or symmetric.

Asymmetric bispecific antibodies generally comprise a heavy chain/lightchain (HC/LC) pair from an antibody specific for target A and an HC/LCpair from an antibody specific for target B, creating ahetero-bifunctional antibody. Hetero-bifunctional antibodies such asthese face the problem of unproductive formation of the molecule when itis being produced. HC/LC-A:HC/LC-B is desired, but is usuallythermodynamically or statistically unfavorable from all the possiblecombinations possible. Multiple schemes have been introduced tocircumvent this problem. In some instances, the HC/LC pair from anantibody with specificity for A and the HC/LC pair from an antibody withspecificity for B further comprise mutations to the FC region toincrease the probability of formation of an antibody withHC/LC-A:HC/LC-B. This can be achieved by engineering structural featuressuch as “knobs” into the FC region for HC-A, and “holes” into HC-B, orvice versa, that promote formation of heterodimers between HC-A andHC-B. Another scheme to promote HC-A:HC-B heterodimers is to engineeramino acid residues in the FC portion of HC-A and HC-B to comprisecharge pairs that favor electrostatic interactions between HC-B andHC-A. Another scheme to address the problem of chain association is toreplace the variable regions of one of the HC/LC pairs with asingle-chain binding molecules (e.g., VHH or an scFv). Such thatone-half of the molecule comprises a classical HC/LC pair and the othercomprises a HC constant region fused or otherwise connected to thesingle-chain binding molecule. Further modifications can be made topromote proper HC/LC paring and include engineering mutations to the HCand LC for either A or B to favor formation of the proper HC/LC pair;CrossMab technology, which entails swapping the corresponding constantregions of the HC/LC pair. Symmetric bispecific antibodies circumventthe chain association problem by not relying on formation of ahetero-bifunctional molecule. Such examples include: the dual-variabledomain molecule, which comprises stacked variable regions of differingspecificity; the IgG-scFv molecule, which comprises an scFv of adiffering specificity fused to the c-terminus of heavy chain of aclassical antibody molecule; the (scFV)4-FC, which comprises two scFvsconnected by an Fc region of an Ig (the Fcs dimerize creating abispecific, tetravalent molecule); the DART-Fc and the two-in-one,amongst others.

The structure of composite binding molecules or bispecific antibodiescan be conceived and designed to alter functionality or bindingproperties of the composite binding molecules or bispecific antibodies(see e.g., “Bispecific antibodies: a mechanistic review of thepipeline.” Nat Rev Drug Discovery. 2019 Aug;18(8):585-608) (see e.g.,“The making of bispecific antibodies” MAbs. 2017 Feb-Mar; 9(2):182-212). For example, the bispecific antibody can be selected from oneof the following formats: a common light chain bispecific IgG, aFab-Fc:scFv-Fc bispecific IgG, a Fab-Fc-Fab:Fc bispecific IgG, aFab-Fc-scFv:Fab-Fc-scFv bispecific IgG, a Fab-Fc-scFv:Fc bispecific IgG,a Fab-Fc-Fab:Fab-Fc bispecific IgG, an scFv-Fab-Fc:scFv-Fab-Fcbispecific IgG, a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG, aFab-Fc-Fab:Fab-Fc-Fab bispecific IgG, and a Fab-Fc-scFv:Fab-Fcbispecific IgG.

Common Light Chain Bispecific IgG

A bispecific antibody having a common light chain bispecific IgGstructure can be used herein. FIG. 1 illustrates a bispecific antibodyhaving a common light chain bispecific IgG structure. The structurecomprises a first and a second IgG heavy chain. Each heavy chaincomprises a VH, CH1, CH2, and CH3 domain. The first heavy chaincomprises VH 102, CH1 104, CH2 106, and CH3 108. The second heavy chaincomprises VH 112, CH1 114, CH2 116, and CH3 118. The common light chainbispecific IgG structure also comprises a light chain comprising a VLdomain 120 and a CL domain 122. Generally, the first heavy chain willcomprise a sequence derived from the heavy chain of an antibody with afirst specificity; and the second heavy chain will comprise a heavychain from an antibody with a second specificity. The light chain thatpairs with the first and the second heavy chain will be identical, andcan be derived from the light chain of an antibody with eitherspecificity, or a separate specificity. A heavy chain can be covalentlycoupled to a light chain molecule via a covalent bond (e.g. disulfidebond 130). A heavy chain can be coupled to another heavy chain via oneor more covalent bonds (e.g. disulfide bond 134 and/or 136). The commonlight chain bispecific IgG structure can comprise a first and a secondheavy chain molecule that further comprises mutations within the CH3domain that promote coupling of the first and the second heavy chainand/or prevent coupling of a first heavy chain to another first heavychain or a second heavy chain to another second heavy chain. Themutations can physically (e.g. steric hinderance, “knobs” into “holes”)or biochemically (e.g. electrostatic interactions) prevent coupling ofthe two first heavy chain molecules or two second heavy chain molecules.Exemplary knob into hole mutations can comprise T366W (EU numbering) inone heavy chain and T366S/L368A/Y407V (EU numbering) in a second heavychain. Exemplary mutations that facilitate coupling of a first and asecond heavy chain molecule are disclosed, for example in WO2009089004,US 8,642,745, US PG-PUB: US20140322756 and “The making of bispecificantibodies” MAbs. 2017 Feb-Mar; 9(2): 182-212. The common light chainbispecific IgG structure can also comprise carbohydrate molecules 140coupled thereto or additional modifications thereof.

A bispecific antibody having a common light chain bispecific IgGstructure can target a B-cell lineage surface marker (e.g. CD19, CD138,IgA, or CD45), and a suppressive B-cell surface marker (e.g. IgD, CD1,CDS, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g.,TGF-beta LAP)). In some embodiments, the first heavy chain is configuredto bind B-cell lineage surface marker and the second heavy is configuredto bind a suppressive B-cell surface marker. In some embodiments, theB-cell lineage surface marker comprises CD19. In certain embodiments,the B-cell lineage surface marker consists of CD19. In some embodiments,the suppressive B-cell surface marker comprises CD38. In certainembodiments, the suppressive B-cell surface marker consists of CD38.

In some embodiments, the first heavy chain comprises a VH sequencecomprising a CD19 binding component and the second heavy chain comprisesa VH sequence comprising CD38 binding component. In certain embodiments,the heavy chain CD19 binding component comprises SEQ ID NO: 201, 1, or avariant comprising a mutation at one or both of A84 and A108 of SEQ IDNO: 201 and the heavy chain CD38 binding component comprises SEQ ID NOs:202, 215, 218-221. In certain embodiments, the variant comprises themutation A84S and A108L. In some embodiments, the bispecific antibodycomprises a common light chain. In certain embodiments, the common lightchain sequence comprises a CD19 binding component (e.g. SEQ ID NO: 2).In certain embodiments, the common light chain sequence comprises CD38binding component (e.g. SEQ ID NO: 4 or SEQ ID NO: 222).

Described herein BS1 comprises a common light chain format with a CD19binding component configured to bind CD19 and a CD38 binding componentconfigured to bind CD38, wherein the CD19 binding component comprises anantibody or antigen binding fragment thereof and the CD38 bindingcomponent comprises an antibody or antigen binding fragment thereof,wherein the CD38 antibody or antigen binding fragment comprises ananti-CD38 immunoglobulin heavy chain variable region paired with ananti-CD38 immunoglobulin light chain variable region and the CD19antibody or antigen binding fragment comprises an anti-CD19immunoglobulin heavy chain variable region paired with an anti-CD38immunoglobulin light chain variable region, wherein the CD38 antibody orantigen binding component comprises: a) a heavy chain complementaritydetermining region 1 (HCDR1) comprising an amino acid sequence set forthin any one of SEQ ID NOs: 71-75, b) a heavy chain complementaritydetermining region 2 (HCDR2) comprising an amino acid sequence set forthin any one of SEQ ID NOs: 81-85, or 150-155; c) a heavy chaincomplementarity determining region 3 (HCDR3) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 91-95; d) a light chaincomplementarity determining region 1 (LCDR1) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 101-105;e) a light chaincomplementarity determining region 2 (LCDR2) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 111-115; and/or) a lightchain complementarity determining region 3 (LCDR3) comprising an aminoacid sequence set forth in any one of SEQ ID NOs: 121-125; and whereinthe CD19 antigen binding component comprises: g) a heavy chaincomplementarity determining region 1 (HCDR1) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 11-15, h) a heavy chaincomplementarity determining region 2 (HCDR2) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 21-25, i) a heavy chaincomplementarity determining region 3 (HCDR3) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 31-35; j) a light chaincomplementarity determining region 1 (LCDR1) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 101-105; k) a light chaincomplementarity determining region 2 (LCDR2) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 111-115; and/or 1) a lightchain complementarity determining region 3 (LCDR3) comprising an aminoacid sequence set forth in any one of SEQ ID NOs: 121-125. In someembodiments, the CD 38 antigen binding component comprises a HCDR2 aminoacid sequence comprising the sequence P-X1-L-G-X2-A (SEQ ID NO: 156),wherein X1 and X2 are each selected from the group consisting of H, Q,T, N, S, G, A, R, K, D, or E. In certain embodiments, the X1 is H and X2is T. In some embodiments, the CD19 heavy chain sequence comprises aA84S and/or A108L substitution. In some embodiments, the CD38 lightchain comprises a W32H substitution.

Fab-Fc:scFv-Fc Bispecific IgG

A bispecific antibody having a Fab-Fc:scFv-Fc Bispecific IgG structurecan be used herein. FIG. 2 illustrates a bispecific antibody having aFab-Fc:scFv-Fc Bispecific IgG structure. The structure comprises a firstheavy chain molecule and a modified second IgG heavy chain moleculecomprising a single chain variable fragment. The first heavy chaincomprises VH 202, CH1 204, CH2 206, and CH3 208, N-terminus toC-terminus respectively. The modified second heavy chain comprises asingle chain variable fragment (scFv) 210, CH2 216, and CH3 218,N-terminus to C-terminus respectively. The single chain variablefragment (scFv) can comprises a first domain 212 corresponding to avariable light chain domain, or fragment thereof, a second domain 214corresponding to a variable heavy chain, or a fragment thereof, and alinker polypeptide 215. The Fab-Fc:scFv-Fc Bispecific IgG structure alsocomprises a light chain comprising a VL domain 220 and a CL domain 222.The first heavy chain can be covalently coupled to a light chainmolecule via a covalent bond (e.g. disulfide bond 230). A first heavychain can be coupled to the modified second heavy chain via one or morecovalent bonds (e.g. disulfide bond 234 and/or 236). The Fab-Fc:scFv-FcBispecific IgG structure can comprise a first and a modified secondheavy chain molecule that further comprises mutations within the CH3domain that promote coupling of the first and the second heavy chainand/or prevent coupling of a first heavy chain to another first heavychain or a second heavy chain to another second heavy chain. Themutations can physically (e.g. steric hinderance) or biochemically (e.g.electrostatic interactions) prevent coupling of the two first heavychain molecules or two second heavy chain molecules. Exemplary mutationsthat facilitate coupling of a first and a second heavy chain moleculeare disclosed, for example in US PG-PUB: US20140322756 and “The makingof bispecific antibodies” MAbs. 2017 Feb-Mar; 9(2): 182-212. TheFab-Fc:scFv-Fc Bispecific IgG structure can also comprise carbohydratemolecules 240 coupled thereto or additional modifications thereof.

A bispecific antibody having a Fab-Fc:scFv-Fc Bispecific IgG structurecan target a B-cell lineage surface marker (e.g. CD19, CD138, IgA, orCD45e.g. CD19, CD138, IgA, or CD45), and a suppressive B-cell surfacemarker (e.g. IgD, CD1, CDS, CD21, CD24, CD38, HM13, SLAMF7, AQP3, orlatent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B-celllineage surface marker comprises CD19. In certain embodiments, theB-cell lineage surface marker consists of CD19. In some embodiments, thesuppressive B-cell surface marker comprises CD38. In certainembodiments, the suppressive B-cell surface marker consists of CD38.

The Fab-Fc:scFv-Fc Bispecific IgG structure can be engineered so that afirst antigen binding site targets CD19 and a second antigen bindingsite targets CD38. In some embodiments, the first heavy chain comprisesa VH sequence comprising CD19 binding component and the second heavychain comprises a single chain variable fragment (scFv) sequencecomprising a CD38 binding component. In certain embodiments, the heavychain comprising the CD38 single chain variable fragment comprises SEQID NO: 205 or SEQ ID NO: 206. In certain embodiments, the VL sequencecomprises a CD19 binding component. In certain embodiments, the singlechain variable fragment (scFv) sequence comprising a CD38 bindingcomponent comprises a CD38 binding component corresponding to anantibody heavy chain and light variable sequence, or CD38 bindingfragment thereof. In some embodiments, the first heavy chain comprises aVH sequence comprising CD38 binding component and the second heavy chaincomprises a single chain variable fragment (scFv) sequence comprising aCD19 binding component. In certain embodiments, the heavy chaincomprising the CD19 single chain variable fragment comprises SEQ ID NO:203 or SEQ ID NO: 204 or SEQ ID NO: 217. In certain embodiments, thesingle chain variable fragment (scFv) sequence comprising a CD19 bindingcomponent comprises a CD19 binding component corresponding to anantibody heavy chain and light variable sequence, or CD19 bindingfragment thereof.

The Fab-Fc:scFv-Fc Bispecific IgG structure can be engineered so that afirst antigen binding site targets CD38 and a second antigen bindingsite targets CD19. In some embodiments, the first heavy chain comprisesa VH sequence comprising CD38 binding component and the second heavychain comprises a single chain variable fragment (scFv) sequencecomprising a CD19 binding component. In certain embodiments, the VLsequence comprises a CD38 binding component. In certain embodiments, thesingle chain variable fragment (scFv) sequence comprising a CD19 bindingcomponent comprises a CD19 binding component corresponding to anantibody heavy chain and light variable sequence, or CD19 bindingfragment thereof.

Described herein BS2 comprises a CD19 binding component configured tobind CD19 and a CD38 binding component configured to bind CD38, whereinthe CD19 binding component comprises an antibody or antigen bindingfragment thereof and the CD38 binding component comprises an antibody orantigen binding fragment thereof, wherein the CD38 antigen bindingcomponent comprises a Fab that binds CD38 comprising an anti-CD38immunoglobulin heavy chain variable region paired with an anti-CD38immunoglobulin light chain variable region and the CD19 antigen bindingcomponent comprises an scFv that binds CD19 comprising an anti-CD19immunoglobulin heavy chain variable region paired with an anti-CD38immunoglobulin light chain variable region, wherein the CD 38 bindingcomponent comprises an immunoglobulin heavy chain comprising an HCDR1amino acid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or150-155, an HCDR3 amino acid sequence set forth in any one of SEQ IDNOs: 91-95; and the immunoglobulin light chain comprises an LCDR1 aminoacid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and/oran LCDR3 amino acid sequence set forth in any one of SEQ ID NOs:121-125; and wherein the CD 19 binding component comprises animmunoglobulin heavy chain comprising an HCDR1 amino acid sequence setforth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence setforth in any one of SEQ ID NOs: 21-25, an HCDR3 amino acid sequence setforth in any one of SEQ ID NOs: 31-35; and the immunoglobulin lightchain comprises an LCDR1 amino acid sequence set forth in any one of SEQID NOs: 41-45, an LCDR2 amino acid sequence set forth in any one of SEQID NOs: 51-55, and/or an LCDR3 amino acid sequence set forth in any oneof SEQ ID NOs: 61-65.In some embodiments, the CD 38 antigen bindingcomponent comprises a HCDR2 amino acid sequence comprising the sequenceP-X1-L-G-X2-A (SEQ ID NO: 156), wherein X1 and X2 are selected from thegroup consisting of H, Q, T, N, S, G, A, R, K, D, or E. In certainembodiments, the X1 is H and X2 is T. In some embodiments, the CD19heavy chain sequence comprises a A84S and/or A108L substitution. In someembodiments, the CD38 light chain comprises a W32H substitution.

Fab-Fc-Fab:Fc Bispecific IgG

An engineered bispecific antibody having a Fab-Fc-Fab:Fc Bispecific IgGstructure can be used herein. FIG. 3 illustrates a bispecific antibodyhaving a Fab-Fc-Fab:Fc Bispecific IgG structure. The structure comprisesa first heavy chain molecule and a modified IgG heavy chain molecule.The first heavy chain comprises VH domain 302, CH1 domain 304, CH2domain 306, CH3 domain 308, a linker 310, a second VH domain 312, and asecond CH1 domain 314, N-terminus to C-terminus respectively. Themodified heavy chain comprises a CH2 domain 316, and CH3 domain 318,N-terminus to C-terminus respectively. The Fab-Fc-Fab:Fc Bispecific IgGstructure also comprises a first light chain comprising a VL domain 320and a CL domain 322. The Fab-Fc-Fab:Fc Bispecific IgG structure alsocomprises a second light chain comprising a VL domain 324 and a CLdomain 326. A heavy chain can be covalently coupled to a light chainmolecule via a covalent bond (e.g. disulfide bond 330). The first heavychain can also be covalently coupled to the first second chain moleculevia a covalent bond (e.g. disulfide bond 332). A heavy chain and a lightchain can be coupled in a manner that the VH domain and CH1 domain ofthe first heavy chain pair with the VL domain and CL domain of the firstlight chain. The first heavy chain and second light chain can be coupledin a manner that the second VH domain and second CH1 domain of the firstheavy chain pair with the VL domain and CL domain of the second lightchain. The first heavy chain can be coupled to the modified second heavychain via one or more covalent bonds (e.g. disulfide bond 334 and/or336). The Fab-Fc-Fab:Fc Bispecific IgG structure can comprise a firstand a modified second heavy chain molecule that further comprisesmutations within the CH3 domain that promote coupling of the first andthe second heavy chain and/or prevent coupling of a first heavy chain toanother first heavy chain or a second heavy chain to another secondheavy chain. The mutations can physically (e.g. steric hinderance) orbiochemically (e.g. electrostatic interactions) prevent coupling of thetwo first heavy chain molecules or two second heavy chain molecules.Exemplary mutations that facilitate coupling of a first and a secondheavy chain molecule are disclosed, for example in US PG-PUB:US20140322756 and “The making of bispecific antibodies” MAbs. 2017Feb-Mar; 9(2): 182-212. The Fab-Fc-Fab:Fc Bispecific IgG structure canalso comprise carbohydrate molecules 340 coupled thereto or additionalmodifications thereof

A bispecific antibody having a Fab-Fc-Fab:Fc Bispecific IgG structurecan target a B-cell lineage surface marker (e.g. CD19, CD138, IgA, orCD45e.g. CD19, CD138, IgA, or CD45), and a suppressive B-cell surfacemarker (e.g. IgD, CD1, CDS, CD21, CD24, CD38, HM13, SLAMF7, AQP3, orlatent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B-celllineage surface marker comprises CD19. In certain embodiments, theB-cell lineage surface marker consists of CD19. In some embodiments, thesuppressive B-cell surface marker comprises CD38. In certainembodiments, the suppressive B-cell surface marker consists of CD38.

The Fab-Fc-Fab:Fc Bispecific IgG structure can be engineered so that afirst antigen binding site targets CD19 and a second antigen bindingsite targets CD38. In some embodiments, the first heavy chain VH domain(e.g. 302) and VL domain (e.g. 320) comprises a CD19 binding component,wherein the second VH domain (e.g. 312) and VL domain (e.g. 324)comprises a CD38 binding component. In some embodiments, the Fab-Fc-Fabheavy chain comprises SEQ ID NO: 207 and the Fc heavy chain comprisesSEQ ID NO: 208.

The Fab-Fc-Fab:Fc Bispecific IgG structure can also be engineered sothat a first antigen binding site targets CD38 and a second antigenbinding site targets CD19. In some embodiments, the first heavy chain VHdomain (e.g. 302) and VL domain (e.g. 320) comprises a CD38 bindingcomponent, wherein the second VH domain (e.g. 312) and VL domain (e.g.324) comprises a CD19 binding component.

Fab-Fc-scFv:Fab-Fc-scFv Bispecific IgG

An engineered bispecific antibody having a Fab-Fc-scFv:Fab-Fc-scFvBispecific IgG structure can be used herein. FIG. 4 illustrates abispecific antibody having a Fab-Fc-scFv:Fab-Fc-scFv Bispecific IgGstructure. The structure comprises a two first heavy chain molecules.The first heavy chain comprises VH domain 402, CH1 domain 404, CH2domain 406, CH3 domain 408, a linker 410, and a single chain variablefragment (scFv) 412, N-terminus to C-terminus respectively. The singlechain variable fragment (scFv) can comprises a first domain 414corresponding to a variable light chain domain, or fragment thereof, asecond domain 416 corresponding to a variable heavy chain, or a fragmentthereof, and a second linker polypeptide 415. TheFab-Fc-scFv:Fab-Fc-scFv Bispecific IgG structure also comprises a firstlight chain comprising a VL domain 420 and a CL domain 422. A heavychain can be covalently coupled to a light chain molecule via a covalentbond (e.g. disulfide bond 430). A heavy chain can be coupled to anotherheavy chain via one or more covalent bonds (e.g. disulfide bond 434and/or 436). The Fab-Fc-scFv:Fab-Fc-scFv Bispecific IgG structure canalso comprise carbohydrate molecules 440 coupled thereto or additionalmodifications thereof.

A bispecific antibody having a Fab-Fc-scFv:Fab-Fc-scFv Bispecific IgGstructure can target a B-cell lineage surface marker (e.g. CD19, CD138,IgA, or CD45e.g. CD19, CD138, IgA, or CD45), and a suppressive B-cellsurface marker (e.g. IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7,AQP3, or latent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, theB-cell lineage surface marker comprises CD19. In certain embodiments,the B-cell lineage surface marker consists of CD19. In some embodiments,the suppressive B-cell surface marker comprises CD38. In certainembodiments, the suppressive B-cell surface marker consists of CD38.

The Fab-Fc-scFv:Fab-Fc-scFv Bispecific IgG structure can be engineeredso that a first antigen binding site targets CD19 and a second antigenbinding site targets CD38. In some embodiments, the first heavy chain VHdomain (e.g. 402) and VL domain (e.g. 420) comprises a CD19 bindingcomponent, wherein the single chain variable fragment (scFv) (e.g. 412)sequence comprises a CD38 binding component. In certain embodiments, thesingle chain variable fragment (scFv) sequence comprising a CD38 bindingcomponent comprises a CD38 binding component corresponding to anantibody heavy chain and light variable sequence, or CD38 bindingfragments thereof

The Fab-Fc-scFv:Fab-Fc-scFv Bispecific IgG structure can also beengineered so that a first antigen binding site targets CD38 and asecond antigen binding site targets CD19. In some embodiments, the firstheavy chain VH domain (e.g. 402) and VL domain (e.g. 420) comprises aCD38 binding component, wherein the single chain variable fragment(scFv) (e.g. 412) sequence comprises a CD19 binding component. Incertain embodiments, the single chain variable fragment (scFv) sequencecomprising a CD19 binding component comprises a CD19 binding componentcorresponding to an antibody heavy chain and light variable sequence, orCD19 binding fragments thereof In some embodiments, the Fab-Fc-scFvheavy chain comprises SEQ ID NO: 209.

Fab-Fc-scFv:Fc Bispecific IgG

An engineered bispecific antibody having a Fab-Fc-scFv:Fc Bispecific IgGstructure can be used herein. FIG. 5 illustrates a bispecific antibodyhaving a Fab-Fc-scFv:Fc Bispecific IgG structure. The structurecomprises a first heavy chain molecule and a second IgG heavy chainmolecule. The first heavy chain comprises VH domain 502, CH1 domain 504,CH2 domain 506, CH3 domain 508, a linker 510, and a single chainvariable fragment (scFv) 512, N-terminus to C-terminus respectively. Thesingle chain variable fragment (scFv) can comprises a first domain 514corresponding to a variable light chain domain, or fragment thereof, asecond domain 516 corresponding to a variable heavy chain, or a fragmentthereof, and a second linker polypeptide 515. The Fab-Fc-scFv:FcBispecific IgG structure also comprises a first light chain comprising aVL domain 520 and a CL domain 522. The Fab-Fc-scFv:Fc Bispecific IgGstructure also comprises a second light chain comprising a VL domain 524and a CL domain 526. A heavy chain can be covalently coupled to a lightchain molecule via a covalent bond (e.g. disulfide bond 530). A heavychain can be coupled to another heavy chain via one or more covalentbonds (e.g. disulfide bond 534 and/or 536). The Fab-Fc-scFv:FcBispecific IgG structure can comprise a first and a modified secondheavy chain molecule that further comprises mutations within the CH3domain that promote coupling of the first and the second heavy chainand/or prevent coupling of a first heavy chain to another first heavychain or a second heavy chain to another second heavy chain. Themutations can physically (e.g. steric hinderance) or biochemically (e.g.electrostatic interactions) prevent coupling of the two heavy chainmolecules or two second heavy chain molecules. Exemplary mutations thatfacilitate coupling of a first and a second heavy chain molecule aredisclosed, for example in US PG-PUB: US20140322756 and “The making ofbispecific antibodies” MAbs. 2017 Feb-Mar; 9(2): 182-212. TheFab-Fc-scFv:Fc Bispecific IgG structure can also comprise carbohydratemolecules 540 coupled thereto or additional modifications thereof.

A bispecific antibody having a Fab-Fc-scFv:Fc Bispecific IgG structurecan target a B-cell lineage surface marker (e.g. CD19, CD138, IgA, orCD45e.g. CD19, CD138, IgA, or CD45), and a suppressive B-cell surfacemarker (e.g. IgD, CD1, CDS, CD21, CD24, CD38, HM13, SLAMF7, AQP3, orlatent TGF-beta (e.g., TGF-beta LAP)). In some embodiments, the B-celllineage surface marker comprises CD19. In certain embodiments, theB-cell lineage surface marker consists of CD19. In some embodiments, thesuppressive B-cell surface marker comprises CD38. In certainembodiments, the suppressive B-cell surface marker consists of CD38.

The Fab-Fc-scFv:Fc Bispecific IgG structure can be engineered so that afirst antigen binding site targets CD19 and a second antigen bindingsite targets CD38. In some embodiments, the first heavy chain VH domain(e.g. 502) and VL domain (e.g. 520) comprises a CD19 binding component,wherein the single chain variable fragment (scFv) (e.g. 512) sequencecomprises a CD38 binding component. In certain embodiments, the singlechain variable fragment (scFv) sequence comprising a CD38 bindingcomponent comprises a CD38 binding component corresponding to anantibody heavy chain and light variable sequence, or CD38 bindingfragments thereof.

The Fab-Fc-scFv:Fc Bispecific IgG structure can also be engineered sothat a first antigen binding site targets CD38 and a second antigenbinding site targets CD19. In some embodiments, the first heavy chain VHdomain (e.g. 502) and VL domain (e.g. 520) comprises a CD38 bindingcomponent, wherein the single chain variable fragment (scFv) (e.g. 512)sequence comprises a CD19 binding component. In certain embodiments, thesingle chain variable fragment (scFv) sequence comprising a CD19 bindingcomponent comprises a CD19 binding component corresponding to anantibody heavy chain and light variable sequence, or CD19 bindingfragments thereof.

Fab-Fc-Fab:Fab-Fc Bispecific IgG

An engineered bispecific antibody having a Fab-Fc-Fab:Fab-Fc BispecificIgG structure can be used herein. FIG. 6 illustrates a bispecificantibody having a Fab-Fc-Fab:Fab-Fc Bispecific IgG structure. Thestructure comprises a first heavy chain molecule and a second IgG heavychain molecule. The first heavy chain comprises VH domain 602, CH1domain 604, CH2 domain 606, CH3 domain 608, a linker 610 a second VHdomain 612, and a second CH1 domain 614, N-terminus to C-terminusrespectively. The second heavy chain comprises a VH domain 652, a CH1domain 654, a CH2 domain 656, and CH3 domain 658, N-terminus toC-terminus respectively, as in that of the first heavy chain. TheFab-Fc-Fab:Fab-Fc Bispecific IgG structure also comprises a first lightchain comprising a VL domain 620 and a CL domain 622. TheFab-Fc-Fab:Fab-Fc Bispecific IgG structure also comprises a second lightchain comprising a VL domain 624 and a CL domain 626. A heavy chain canbe covalently coupled to a light chain molecule via a covalent bond(e.g. disulfide bond 630). The first heavy chain and first light chaincan be coupled in a manner that the VH domain and CH1 domain of thefirst heavy chain pair with the VL domain and CL domain of the firstlight chain. The first heavy chain and second light chain can be coupledin a manner that the second VH domain and second CH1 domain of the firstheavy chain pair with the VL domain and CL domain of the second lightchain. A heavy chain can be coupled to another heavy chain via one ormore covalent bonds (e.g. disulfide bond 634 and/or 636). TheFab-Fc-Fab:Fab-Fc Bispecific IgG structure can comprise a first and asecond heavy chain molecule that further comprises mutations within theCH3 domain that promote coupling of the first and the second heavy chainand/or prevent coupling of a first heavy chain to another first heavychain or a second heavy chain to another second heavy chain. Themutations can physically (e.g. steric hinderance) or biochemically (e.g.electrostatic interactions) prevent coupling of the two first heavychain molecules or two second heavy chain molecules. Exemplary mutationsthat facilitate coupling of a first and a second heavy chain moleculeare disclosed, for example in US PG-PUB: US20140322756 and “The makingof bispecific antibodies” MAbs. 2017 Feb-Mar; 9(2): 182-212. TheFab-Fc-Fab:Fab-Fc Bispecific IgG structure can also comprisecarbohydrate molecules coupled thereto or additional modificationsthereof.

A bispecific antibody having a Fab-Fc-Fab:Fab-Fc Bispecific IgGstructure can target a B-cell lineage surface marker (e.g. CD19, CD138,IgA, or CD45), and a suppressive B-cell surface marker (e.g. IgD, CD1,CDS, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g.,TGF-beta LAP)). In some embodiments, the B-cell lineage surface markercomprises CD19. In certain embodiments, the B-cell lineage surfacemarker consists of CD19. In some embodiments, the suppressive B-cellsurface marker comprises CD38. In certain embodiments, the suppressiveB-cell surface marker consists of CD38.

The Fab-Fc-Fab:Fab-Fc Bispecific IgG structure can be engineered so thata first antigen binding site targets CD19 and a second antigen bindingsite targets CD38. In some embodiments, the first heavy chain VH domain(e.g. 602) and VL domain (e.g. 620) comprises a CD19 binding component,wherein the second VH domain (e.g. 612) and VL domain (e.g. 624)comprises a CD38 binding component.

The Fab-Fc-Fab:Fab-Fc Bispecific IgG structure can also be engineered sothat a first antigen binding site targets CD38 and a second antigenbinding site targets CD19. In some embodiments, the first heavy chain VHdomain (e.g. 602) and VL domain (e.g. 620) comprises a CD38 bindingcomponent, wherein the second VH domain (e.g. 612) and VL domain (e.g.624) comprises a CD19 binding component.

scFv-Fab-Fc:scFv-Fab-Fc Bispecific IgG

An engineered bispecific antibody having an scFv-Fab-Fc:scFv-Fab-FcBispecific IgG structure can be used herein. FIG. 7 illustrates abispecific antibody having an scFv-Fab-Fc:scFv-Fab-Fc Bispecific IgGstructure. The structure comprises a two first heavy chain molecules.The first heavy chain comprises a single chain variable fragment (scFv)712, a linker 710, VH domain 702, CH1 domain 704, CH2 domain 706, and aCH3 domain 708, N-terminus to C-terminus respectively. The single chainvariable fragment (scFv) can comprises a first domain 714 correspondingto a variable light chain domain, or fragment thereof, a second domain716 corresponding to a variable heavy chain, or a fragment thereof, anda second linker polypeptide 715. The ScFv-Fab-Fc:scFv-Fab-Fc BispecificIgG structure also comprises a first light chain comprising a VL domain720 and a CL domain 722. A heavy chain can be covalently coupled to alight chain molecule via a covalent bond (e.g. disulfide bond 730). Aheavy chain can be coupled to another heavy chain via one or morecovalent bonds (e.g. disulfide bond 734 and/or 736). TheScFv-Fab-Fc:scFv-Fab-Fc Bispecific IgG structure can also comprisecarbohydrate molecules 740 coupled thereto or additional modificationsthereof.

A bispecific antibody having an scFv-Fab-Fc:scFv-Fab-Fc Bispecific IgGstructure can target a B-cell lineage surface marker (e.g. CD19, CD138,IgA, or CD45), and a suppressive B-cell surface marker (e.g. IgD, CD1,CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g.,TGF-beta LAP)). In some embodiments, the B-cell lineage surface markercomprises CD19. In certain embodiments, the B-cell lineage surfacemarker consists of CD19. In some embodiments, the suppressive B-cellsurface marker comprises CD38. In certain embodiments, the suppressiveB-cell surface marker consists of CD38.

The scFv-Fab-Fc:scFv-Fab-Fc Bispecific IgG structure can be engineeredso that a first antigen binding site targets CD19 and a second antigenbinding site targets CD38. In some embodiments, the first heavy chain VHdomain (e.g. 702) and VL domain (e.g. 720) comprises a CD19 bindingcomponent, wherein the single chain variable fragment (scFv) (e.g. 712)sequence comprises a CD38 binding component. In certain embodiments, thesingle chain variable fragment (scFv) sequence comprising a CD38 bindingcomponent comprises a CD38 binding component corresponding to anantibody heavy chain and light variable sequence, or CD38 bindingfragments thereof

The scFv-Fab-Fc:scFv-Fab-Fc Bispecific IgG structure can also beengineered so that a first antigen binding site targets CD38 and asecond antigen binding site targets CD19. In some embodiments, the firstheavy chain VH domain (e.g. 702) and VL domain (e.g. 720) comprises aCD38 binding component, wherein the single chain variable fragment(scFv) (e.g. 712) sequence comprises a CD19 binding component. Incertain embodiments, the single chain variable fragment (scFv) sequencecomprising a CD19 binding component comprises a CD19 binding componentcorresponding to an antibody heavy chain and light variable sequence, orCD19 binding fragments thereof.

Fab-Fab-Fc:Fab-Fab-Fc Bispecific IgG

An engineered bispecific antibody having a Fab-Fab-Fc:Fab-Fab-FcBispecific IgG structure can be used herein. FIG. 8 illustrates abispecific antibody having a Fab-Fab-Fc:Fab-Fab-Fc Bispecific IgGstructure. The structure comprises two heavy chain molecules. The heavychain comprises an additional VH domain 812, and an additional CH1domain 814, a linker 810, VH domain 802, CH1 domain 804, CH2 domain 806,and a CH3 domain 808, N-terminus to C-terminus respectively. TheFab-Fab-Fc:Fab-Fab-Fc Bispecific IgG structure also comprises a firstlight chain comprising a VL domain 820 and a CL domain 822. TheFab-Fab-Fc:Fab-Fab-Fc Bispecific IgG structure also comprises a secondlight chain comprising a VL domain 824 and a CL domain 826. A heavychain molecule can be covalently coupled to a light chain molecule via acovalent bond (e.g. disulfide bond 830). The heavy chain and first lightchain can be coupled in a manner that the VH domain and CH1 domain ofthe heavy chain pair with the VL domain and CL domain of the first lightchain. The heavy chain and second light chain can be coupled in a mannerthat the additional VH domain and additional CH1 domain of the heavychain pair with the VL domain and CL domain of the second light chain. Aheavy chain can be coupled to the modified second heavy chain via one ormore covalent bonds (e.g. disulfide bond 834 and/or 836). TheFab-Fab-Fc:Fab-Fab-Fc Bispecific IgG structure can also comprisecarbohydrate molecules 840 coupled thereto or additional modificationsthereof.

A bispecific antibody having a Fab-Fab-Fc:Fab-Fab-Fc Bispecific IgGstructure can target a B-cell lineage surface marker (e.g. CD19, CD138,IgA, or CD45), and a suppressive B-cell surface marker (e.g. IgD, CD1,CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g.,TGF-beta LAP)). In some embodiments, the B-cell lineage surface markercomprises CD19. In certain embodiments, the B-cell lineage surfacemarker consists of CD19. In some embodiments, the suppressive B-cellsurface marker comprises CD38. In certain embodiments, the suppressiveB-cell surface marker consists of CD38.

The Fab-Fab-Fc:Fab-Fab-Fc Bispecific IgG structure can be engineered sothat a first antigen binding site targets CD19 and a second antigenbinding site targets CD38. In some embodiments, the first VH domain(e.g. 802) and VL domain (e.g. 820) comprise a CD19 binding component,wherein the second VH domain (e.g. 812) and VL domain (e.g. 824)comprises a CD38 binding component.

The Fab-Fab-Fc:Fab-Fab-Fc Bispecific IgG structure can also beengineered so that a first antigen binding site targets CD38 and asecond antigen binding site targets CD19. In some embodiments, the VHdomain (e.g. 802) and VL domain (e.g. 820) comprises a CD38 bindingcomponent, wherein the second VH domain (e.g. 812) and VL domain (e.g.824) comprises a CD19 binding component.

Fab-Fc-Fab:Fab-Fc-Fab Bispecific IgG

An engineered bispecific antibody having a Fab-Fc-Fab:Fab-Fc-FabBispecific IgG structure can be used herein. FIG. 9 illustrates abispecific antibody having a Fab-Fc-Fab:Fab-Fc-Fab Bispecific IgGstructure. The structure comprises two heavy chain molecules and twolight chain molecules. The heavy chain comprises VH domain 902, CH1domain 904, CH2 domain 906, CH3 domain 908, a linker 910 a second VHdomain 912, and a second CH1 domain 914, N-terminus to C-terminusrespectively. The Fab-Fc-Fab:Fab-Fc-Fab Bispecific IgG structure alsocomprises a first light chain comprising a VL domain 920 and a CL domain922. The Fab-Fc-Fab:Fab-Fc-Fab Bispecific IgG structure also comprises asecond light chain comprising a VL domain 924 and a CL domain 926. Aheavy chain can be covalently coupled to a light chain molecule via acovalent bond (e.g. disulfide bond 930). The heavy chain and first lightchain can be coupled in a manner that the VH domain and CH1 domain ofthe heavy chain pair with the VL domain and CL domain of the first lightchain. The heavy chain and second light chain can be coupled in a mannerthat the second VH domain and second CH1 domain of the heavy chain pairwith the VL domain and CL domain of the second light chain. A heavychain can also be covalently coupled to another heavy chain molecule viaa covalent bond (e.g. disulfide bond 934 and 936). The Fab-Fc-FabBispecific IgG structure can also comprise carbohydrate molecules 940coupled thereto or additional modifications thereof.

A bispecific antibody having a Fab-Fc-Fab:Fab-Fc-Fab Bispecific IgGstructure can target a B-cell lineage surface marker (e.g. CD19, CD138,IgA, or CD45), and a suppressive B-cell surface marker (e.g. IgD, CD1,CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g.,TGF-beta LAP)). In some embodiments, the B-cell lineage surface markercomprises CD19. In certain embodiments, the B-cell lineage surfacemarker consists of CD19. In some embodiments, the suppressive B-cellsurface marker comprises CD38. In certain embodiments, the suppressiveB-cell surface marker consists of CD38.

The Fab-Fc-Fab:Fab-Fc-Fab Bispecific IgG structure can be engineered sothat a first antigen binding site targets CD19 and a second antigenbinding site targets CD38. In some embodiments, the first VH domain(e.g. 902) and VL domain (e.g. 920) comprise a CD19 binding component,wherein the second VH domain (e.g. 912) and VL domain (e.g. 924)comprises a CD38 binding component.

The Fab-Fc-Fab:Fab-Fc-Fab Bispecific IgG structure can also beengineered so that a first antigen binding site targets CD38 and asecond antigen binding site targets CD19. In some embodiments, the VHdomain (e.g. 902) and VL domain (e.g. 920) comprises a CD38 bindingcomponent, wherein the second VH domain (e.g. 912) and VL domain (e.g.924) comprises a CD19 binding component.

Fab-Fc-scFv:Fab-Fc Bispecific IgG

An engineered bispecific antibody having a Fab-Fc-scFv:Fab-Fc BispecificIgG structure can be used herein. FIG. 10 demonstrates a bispecificantibody having a Fab-Fc-scFv:Fab-Fc Bispecific IgG structure. Thestructure comprises a first heavy chain molecule and a second IgG heavychain molecule. The first heavy chain comprises VH domain 1002, CH1domain 1004, CH2 domain 1006, CH3 domain 1008, a linker 1010 and asingle chain variable fragment (scFv) 1012, N-terminus to C-terminusrespectively. The single chain variable fragment (scFv) can comprises afirst domain 1014 corresponding to a variable light chain domain, orfragment thereof, a second domain 1016 corresponding to a variable heavychain, or a fragment thereof, and a second linker polypeptide 1015. Thesecond heavy chain comprises a VH domain 1002, a CH1 domain 1004, a CH2domain 1004, and CH3 domain 1008, N-terminus to C-terminus respectively,as in that of the first heavy chain. The Fab-Fc-scFv:Fab-Fc BispecificIgG structure also comprises a first light chain comprising a VL domain1020 and a CL domain 1022. A heavy chain can be covalently coupled to alight chain molecule via a covalent bond (e.g. disulfide bond 1030). Aheavy chain can be coupled to another heavy chain via one or morecovalent bonds (e.g. disulfide bond 1034 and/or 1036). TheFab-Fc-scFv:Fab-Fc Bispecific IgG structure can comprise a first and asecond heavy chain molecule that further comprises mutations within theCH3 domain that promote coupling of the first and the second heavy chainand/or prevent coupling of a first heavy chain to another first heavychain or a second heavy chain to another second heavy chain. Themutations can physically (e.g. steric hinderance) or biochemically (e.g.electrostatic interactions) prevent coupling of the two first heavychain molecules or two second heavy chain molecules. Exemplary mutationsthat facilitate coupling of a first and a second heavy chain moleculeare disclosed, for example in US PG-PUB: US20140322756 and “The makingof bispecific antibodies” MAbs. 2017 Feb-Mar; 9(2): 182-212. TheFab-Fc-scFv:Fab-Fc Bispecific IgG structure can also comprisecarbohydrate molecules 1040 coupled thereto or additional modificationsthereof.

A bispecific antibody having a Fab-Fc-scFv:Fab-Fc Bispecific IgGstructure can target a B-cell lineage surface marker (e.g. CD19, CD138,IgA, or CD45), and a suppressive B-cell surface marker (e.g. IgD, CD1,CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g.,TGF-beta LAP)). In some embodiments, the B-cell lineage surface markercomprises CD19. In certain embodiments, the B-cell lineage surfacemarker consists of CD19. In some embodiments, the suppressive B-cellsurface marker comprises CD38. In certain embodiments, the suppressiveB-cell surface marker consists of CD38.

The Fab-Fc-scFv:Fab-Fc Bispecific IgG structure can be engineered sothat a first antigen binding site targets CD19 and a second antigenbinding site targets CD38. In some embodiments, the first heavy chain VHdomain (e.g. 1002) and VL domain (e.g. 1020) comprises a CD19 bindingcomponent, wherein the single chain variable fragment (scFv) (e.g. 1012)sequence comprises a CD38 binding component. In certain embodiments, thesingle chain variable fragment (scFv) sequence comprising a CD38 bindingcomponent comprises a CD38 binding component corresponding to anantibody heavy chain and light variable sequence, or CD38 bindingfragments thereof

The Fab-Fc-scFv:Fab-Fc Bispecific IgG structure can also be engineeredso that a first antigen binding site targets CD38 and a second antigenbinding site targets CD19. In some embodiments, the first heavy chain VHdomain (e.g. 1002) and VL domain (e.g. 1020) comprises a CD38 bindingcomponent, wherein the single chain variable fragment (scFv) (e.g. 1012)sequence comprises a CD19 binding component. In certain embodiments, thesingle chain variable fragment (scFv) sequence comprising a CD19 bindingcomponent comprises a CD19 binding component corresponding to anantibody heavy chain and light variable sequence, or CD19 bindingfragments thereof.

scFv-Fab-Fc:Fc Bispecific IgG

An engineered bispecific antibody having a scFv-Fab-Fc:Fc Bispecific IgGstructure can be used herein. FIG. 11 demonstrates a bispecific antibodyhaving a scFv-Fab-Fc:Fc Bispecific IgG structure. The structurecomprises a first heavy chain molecule comprising an scFv, VH, and an Fcregion and a second heavy chain molecule comprising an Fc. ThescFv-Fab-Fc:Fc Bispecific IgG structure can comprise a first and asecond heavy chain molecule that further comprises mutations within theCH3 domain that promote coupling of the first and the second heavy chainand/or prevent coupling of a first heavy chain to another first heavychain or a second heavy chain to another second heavy chain. Themutations can physically (e.g. Knob-in hole architecture) orbiochemically (e.g. electrostatic interactions) promote association ofthe first heavy chain molecule to the second heavy chain molecule. ThescFv-Fab-Fc:Fc Bispecific IgG structure comprises a light chain moleculeassociated with the first heavy chain molecule that creates a firstantigen binding site. A second antigen binding site is provided by anscFv fragment coupled to the N-terminal endo of the first heavy chain.Exemplary mutations that facilitate coupling of a first and a secondheavy chain molecule are disclosed, for example in US PG-PUB:US20140322756 and “The making of bispecific antibodies” MAbs. 2017Feb-Mar; 9(2): 182-212. The scFv-Fab-Fc:Fc Bispecific IgG structure canalso comprise carbohydrate molecules 1140 coupled thereto or additionalmodifications thereof.

A bispecific antibody having an scFv-Fab-Fc:Fc Bispecific IgG structurecan target a B-cell lineage surface marker (e.g. CD19, CD138, IgA, orCD45), and a suppressive B-cell surface marker (e.g. IgD, CD1, CDS,CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-betaLAP)). In some embodiments, the B-cell lineage surface marker comprisesCD19. In certain embodiments, the B-cell lineage surface marker consistsof CD19. In some embodiments, the suppressive B-cell surface markercomprises CD38. In certain embodiments, the suppressive B-cell surfacemarker consists of CD38.

The scFv-Fab-Fc:Fc Bispecific IgG structure can be engineered so that afirst antigen binding site targets CD19 and a second antigen bindingsite targets CD38. In some embodiments, the first heavy chain VH domainand VL domain comprises a CD19 binding component, wherein the singlechain variable fragment (scFv) sequence comprises a CD38 bindingcomponent. In certain embodiments, the single chain variable fragment(scFv) sequence comprises a CD38 binding component corresponding to anantibody heavy chain and light variable sequence, or CD38 bindingfragments thereof.

The scFv-Fab-Fc:Fc Bispecific IgG structure can also be engineered sothat a first antigen binding site targets CD38 and a second antigenbinding site targets CD19. In some embodiments, the heavy chain VHdomain and VL domain comprises a CD38 binding component, wherein thesingle chain variable fragment (scFv) sequence comprises a CD19 bindingcomponent. In certain embodiments, the single chain variable fragment(scFv) sequence comprising a CD19 binding component comprises a CD19binding component corresponding to an antibody heavy chain and lightvariable sequence, or CD19 binding fragments thereof.

In certain embodiments, the first heavy chain molecule comprises anamino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identicalto the amino acid sequence set forth in SEQ ID NO: 212. In certainembodiments, the first heavy chain molecule comprises an amino acidsequence identical to the amino acid sequence set forth in SEQ ID NO:212.

In certain embodiments, the light chain molecule comprises an amino acidsequence at least about 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence set forth in SEQ ID NO: 213. In certain embodiments,the light chain molecule comprises an amino acid sequence identical tothe amino acid sequence set forth in SEQ ID NO: 213.

In certain embodiments, the second heavy chain molecule comprises anamino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identicalto the amino acid sequence set forth in SEQ ID NO: 214. In certainembodiments, the first heavy chain molecule comprises an amino acidsequence identical to the amino acid sequence set forth in SEQ ID NO:214.

Framework Region

Mutations or reversions to a germline sequence made within the frameworkregions of the heavy and light chains can be advantageous for improvingthe pharmacokinetic and pharmacodynamic properties of the CD19 and CD38binding molecules described herein. In certain instances, mutations orreversions to a germline sequence made within a of the heavy and/orlight chain improve stability of the CD19 and CD38 binding molecules(e.g. the bispecific antibodies described herein). In certain instances,mutations or reversions to a germline sequence made within a of theheavy and/or light chain reduce immunogenicity of the CD19 and CD38binding molecules (e.g. the bispecific antibodies described herein).Accordingly, in some embodiments, a Framework Region of a heavy chainand/or light chain comprises 1, 2, 3,4 5, 8, or 10 mutations orreversions back to a germline sequence. In some embodiments, theFramework Region of a heavy chain and/or light chain comprises 1mutation or reversion back to a germline sequence to 10 mutations orreversions back to a germline sequence. In some embodiments, theFramework Region of a heavy chain and/or light chain comprises at least1 mutation or reversion back to a germline sequence. In someembodiments, the Framework Region of a heavy chain and/or light chaincomprises at most 10 mutations or reversions back to a germlinesequence. In some embodiments, the Framework Region of a heavy chainand/or light chain comprises 1 mutation or reversion back to a germlinesequence to 2 mutations or reversions back to a germline sequence, 1mutation or reversion back to a germline sequence to 3 mutations orreversions back to a germline sequence, 1 mutation or reversion back toa germline sequence to 4 mutations or reversions back to a germlinesequence, 1 mutation or reversion back to a germline sequence to 5mutations or reversions back to a germline sequence, 1 mutation orreversion back to a germline sequence to 8 mutations or reversions backto a germline sequence, 1 mutation or reversion back to a germlinesequence to 10 mutations or reversions back to a germline sequence, 2mutations or reversions back to a germline sequence to 3 mutations orreversions back to a germline sequence, 2 mutations or reversions backto a germline sequence to 4 mutations or reversions back to a germlinesequence, 2 mutations or reversions back to a germline sequence to 5mutations or reversions back to a germline sequence, 2 mutations orreversions back to a germline sequence to 8 mutations or reversions backto a germline sequence, 2 mutations or reversions back to a germlinesequence to 10 mutations or reversions back to a germline sequence, 3mutations or reversions back to a germline sequence to 4 mutations orreversions back to a germline sequence, 3 mutations or reversions backto a germline sequence to 5 mutations or reversions back to a germlinesequence, 3 mutations or reversions back to a germline sequence to 8mutations or reversions back to a germline sequence, 3 mutations orreversions back to a germline sequence to 10 mutations or reversionsback to a germline sequence, 4 mutations or reversions back to agermline sequence to 5 mutations or reversions back to a germlinesequence, 4 mutations or reversions back to a germline sequence to 8mutations or reversions back to a germline sequence, 4 mutations orreversions back to a germline sequence to 10 mutations or reversionsback to a germline sequence, 5 mutations or reversions back to agermline sequence to 8 mutations or reversions back to a germlinesequence, 5 mutations or reversions back to a germline sequence to 10mutations or reversions back to a germline sequence, or 8 mutations orreversions back to a germline sequence to 10 mutations or reversionsback to a germline sequence. In some embodiments, the Framework Regionof a heavy chain and/or light chain comprises 1 mutation or reversionback to a germline sequence, 2 mutations or reversions back to agermline sequence, 3 mutations or reversions back to a germlinesequence, 4 mutations or reversions back to a germline sequence, 5mutations or reversions back to a germline sequence, 8 mutations orreversions back to a germline sequence, or 10 mutations or reversionsback to a germline sequence. In some embodiments, the CD38 bindingmoiety comprises a heavy chain framework region as set forth in SEQ IDNO: 5. In some embodiments, the CD binding moiety comprises a heavychain framework region as set forth in SEQ ID NO: 6 or 7.

Pharmaceutically Acceptable Excipients, Carriers, And Diluents

Compositions comprising the composite binding molecules of the currentdisclosure are included in a pharmaceutical composition comprising oneor more pharmaceutically acceptable excipients, carriers, and diluents.In certain embodiments, the antibodies of the current disclosure areadministered suspended in a sterile and/or isotonic solution. In certainembodiments, the solution comprises about 0.9% NaCl. In certainembodiments, the solution comprises about 5.0% dextrose. In certainembodiments, the solution further comprises one or more of: buffers, forexample, acetate, citrate, histidine, succinate, phosphate, bicarbonateand hydroxymethylaminomethane (Tris); surfactants, for example,polysorbate 80 (Tween® 80), polysorbate 20 (Tween® 20), and poloxamer188; polyol/disaccharide/polysaccharides, for example, glucose,dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran40; amino acids, for example, glycine or arginine; antioxidants, forexample, ascorbic acid, methionine; or chelating agents, for example,EDTA or EGTA.

Subcutaneous formulations for administration of antibodies can compriseone or more of: buffers, for example, acetate, citrate, histidine,succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris);surfactants, for example, polysorbate 80 (Tween® 80), polysorbate 20(Tween® 20), and poloxamer 188; polyol/disaccharide/polysaccharides, forexample, glucose, dextrose, mannose, mannitol, sorbitol, sucrose,trehalose, and dextran 40; amino acids, for example, glycine orarginine; antioxidants, for example, ascorbic acid, methionine; orchelating agents, for example, EDTA or EGTA. Additionally, a compound ormolecule that relieves pain at the injection site can be included, suchas hyaluronidase, for example at a concentration of from about 2,000U/ml to about 12,000 U/ml.

In certain embodiments, the composite binding molecules of the currentdisclosure are shipped/stored lyophilized and reconstituted beforeadministration. In certain embodiments, lyophilized antibodyformulations comprise a bulking agent such as, mannitol, sorbitol,sucrose, trehalose, dextran 40, or combinations thereof. The lyophilizedformulation can be contained in a vial comprised of glass or othersuitable non-reactive material. The antibodies when formulated, whetherreconstituted or not, can be buffered at a certain pH, generally lessthan 7.0. In certain embodiments, the pH can be between 4.5 and 6.5, 4.5and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.

Also described herein are kits comprising one or more of the compositebinding molecules described herein in a suitable container and one ormore additional components selected from: instructions for use; adiluent, an excipient, a carrier, and a device for administration.

In certain embodiments, described herein is a method of preparing acancer treatment comprising admixing one or more pharmaceuticallyacceptable excipients, carriers, or diluents and a composite bindingmolecule of the current disclosure. In certain embodiments, describedherein is a method of preparing a cancer treatment for storage orshipping comprising lyophilizing one or more antibodies of the currentdisclosure.

Production and Manufacture

The nucleic acids encoding the composite binding molecules (e.g.bispecific antibodies) described herein can be used to infect,transfect, transform, or otherwise render a suitable cell transgenic forthe nucleic acid, thus enabling the production of composite bindingmolecules for commercial or therapeutic uses. Standard cell lines andmethods for the production of antibodies from a large-scale cell cultureare known in the art. See e.g., Li et al., “Cell culture processes formonoclonal antibody production.” Mabs. 2010 September-October; 2(5):466-477.

In certain embodiments, a nucleic acid sequence encodes the compositebinding molecule or bispecific antibodies disclosed herein. In certainembodiments, the polynucleotide sequence encoding the composite bindingmolecule is operatively coupled to a eukaryotic regulatory sequence. Insome embodiments, a cell comprises the nucleic acid sequence.

In some embodiments, a cell comprises a nucleic acid encoding thecomposite binding molecules disclosed herein. In certain embodiments,the cell comprises a prokaryotic cell. In certain embodiments, theprokaryotic cell is an Escherichia coli cell. In certain embodiments,the cell comprises a eukaryotic cell. In certain embodiments, theeukaryotic cell is a Chines Hamster Ovary (CHO) cell, an NSO murinemyeloma cell, or a human PER.C6 cell

In certain embodiments, described herein is a method of making acomposite binding molecule comprising culturing a cell comprising anucleic acid encoding a composite binding molecule under conditions invitro sufficient to allow production and secretion of the compositebinding molecules.

In certain embodiments, described herein, is a master cell bankcomprising: (a) a mammalian cell line comprising a nucleic acid encodingan antibody described herein integrated at a genomic location; and (b) acryoprotectant. In certain embodiments, the cryoprotectant comprisesglycerol. In certain embodiments, the master cell bank comprises: (a) aCHO cell line comprising a nucleic acid encoding a composite bindingmolecule integrated at a genomic location; and (b) a cryoprotectant. Incertain embodiments, the cryoprotectant comprises glycerol. In certainembodiments, the master cell bank is contained in a suitable vial orcontainer able to withstand freezing by liquid nitrogen.

Also described herein are methods of making composite binding moleculesdescribed herein. Such methods comprise incubating a cell or cell-linecomprising a nucleic acid encoding the composite binding molecules in acell culture medium under conditions sufficient to allow for expressionand secretion of the composite binding molecules, and further harvestingthe composite binding molecules from the cell culture medium. Theharvesting can further comprise one or more purification steps to removelive cells, cellular debris, non-composite binding molecules proteins orpolypeptides, undesired salts, buffers, and medium components. Incertain embodiments, the additional purification step(s) includecentrifugation, ultracentrifugation, protein A, protein G, protein A/G,or protein L purification, and/or ion exchange chromatography.

Methods of Use

Suppression of the immune response by immunoregulatory cells canfacilitate tumor growth, migration, and metastasis. Immunosuppression ornegative immune modulation can include processes or pathways that resultin the full or partial reduction of the immune response.Immunosuppression can be systemic or localized to a specific site (e.g.the tumor microenvironment), tissue, or region of a subject's orpatient's body. Although B cells are primarily known as a positiveimmune modulator through the production of antibodies that facilitateneutralization of a pathogen, certain populations of B cells canfunction to suppress or negatively regulate the immune response. Suchpopulations of B cells can be defined by the expression of more than onecell surface biomarkers. Immunosuppressive B cells or B-cell populationscan comprise a B-cell linage surface biomarker and a suppressive B-cellsurface biomarker. The B-cell lineage surface markers can comprise CD19,CD138, IgA, or CD45. B-cell surface markers can comprise IgD, CD1, CD5,CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-betaLAP). Immunosuppressive B cells or immunosuppressive B-cell populationscan function to suppress the immune response by suppressing a diverseset of cell subtypes, including T cells, through the secretion ofanti-inflammatory mediators, such as cytokines. Immunosuppressive Bcells can also function in attenuating the immune response by negativelymodulating lymphoid structures and/or facilitating the conversion of Tcells to regulatory T cells. Thus, disclosed herein are methods fortargeting immunosuppressive B-cell populations to effectively modulate aresponse.

Targeting immunosuppressive B cells or B-cell populations can result inthe immune activation or positive modulation of the immune responseagainst a tumor or tumorigenic cell. Provided herein are methods oftreating an individual afflicted with a cancer or a tumor comprisingadministering to the individual afflicted with the cancer or the tumorthe composite binding molecules disclosed herein. Also provided hereinare methods of reducing immunosuppressive B cells in, adjacent to, orsurrounding a tumor of an individual afflicted with a tumor or cancercomprising administering to the individual afflicted with the tumor orthe cancer the composite binding molecules disclosed herein, therebyreducing immunosuppressive B cells in, adjacent to, or surrounding thetumor. Further disclosed are methods of contacting an immunosuppressiveB cell in a subject with a composite binding molecule, wherein themethod comprises administering the composite binding molecule to thesubject. In certain embodiments, the subject has a tumor or cancer.

The type, subtype, or form of a tumor or cancer can be an importantfactor in treatment strategies and methods. In some embodiments, thecancer or tumor is a hematologic cancer. In some embodiments, the canceror tumor is a solid-tissue cancer. In some embodiments, the cancercomprises breast cancer, prostate cancer, pancreatic cancer, lungcancer, kidney cancer, stomach cancer, esophageal cancer, skin cancer,colorectal cancer, or head and neck cancer.

Immunosuppressive B cells can suppress the anti-tumor immune response.In some embodiments, the tumor or cancer comprise B cells comprising aB-cell linage surface biomarker and a suppressive B-cell surfacebiomarker. The B-cell lineage surface markers can comprise CD19, CD138,IgA, or CD45. B-cell surface markers can comprise IgD, CD1, CD5, CD21,CD24, CD38, HM13, SLAMF7, AQP3, or TGFB. In some embodiments, the B-cellsurface markers comprise CD19 (e.g. CD19+) and CD38 (e.g. CD CD38+). InSome embodiments, the tumor infiltrating B cells or theimmunosuppressive B cells comprise CD19+, CD38+ B cells.

In certain embodiments, disclosed herein, are bispecific antibodiesuseful for the treatment of a cancer or tumor. Treatment refers to amethod that seeks to improve or ameliorate the condition being treated.With respect to cancer, treatment includes, but is not limited to,reduction of tumor volume, reduction in growth of tumor volume, increasein progression-free survival, or overall life expectancy. In certainembodiments, treatment will affect remission of a cancer being treated.In certain embodiments, treatment encompasses use as a prophylactic ormaintenance dose intended to prevent reoccurrence or progression of apreviously treated cancer or tumor. It is understood by those of skillin the art that not all individuals will respond equally or at all to atreatment that is administered, nevertheless these individuals areconsidered to be treated.

In certain embodiments, the cancer or tumor is a solid cancer or tumor.In certain embodiments, the cancer or tumor is a blood cancer or tumor.In certain embodiments, the cancer or tumor comprises breast, heart,lung, small intestine, colon, spleen, kidney, bladder, head, neck,ovarian, prostate, brain, pancreatic, skin, bone, bone marrow, blood,thymus, uterine, testicular, and liver tumors. In certain embodiments,tumors which can be treated with the antibodies of the inventioncomprise adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelialcarcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma,hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma,medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma,rhabdomyosarcoma, sarcoma and/or teratoma. In certain embodiments, thetumor/cancer is selected from the group of acral lentiginous melanoma,actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenomas,adenosarcoma, adenosquamous carcinoma, astrocytic tumors, Bartholingland carcinoma, basal cell carcinoma, bronchial gland carcinoma,capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma,chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrialhyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma,ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia,gastronoma, germ line tumors, glioblastoma, glucagonoma,hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma,hepatic adenomatosis, hepatocellular carcinoma, insulinite,intraepithelial neoplasia, intraepithelial squamous cell neoplasia,invasive squamous cell carcinoma, large cell carcinoma, liposarcoma,lung carcinoma, lymphoblastic leukemia, lymphocytic leukemia,leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelialtumor, nerve sheath tumor, medulloblastoma, medulloepithelioma,mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma,neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovariancarcinoma, papillary serous adenocarcinoma, pituitary tumors,plasmacytoma, pseudosarcoma, prostate carcinoma, pulmonary blastoma,renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serouscarcinoma, squamous cell carcinoma, small cell carcinoma, soft tissuecarcinoma, somatostatin secreting tumor, squamous carcinoma, squamouscell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucouscarcinoma, vagina/vulva carcinoma, VlPpoma, and Wilm's tumor. In certainembodiments, the tumor/cancer to be treated with one or more antibodiesof the invention comprise brain cancer, head and neck cancer, colorectalcarcinoma, acute myeloid leukemia, pre-B-cell acute lymphoblasticleukemia, bladder cancer, astrocytoma, preferably grade II, III or IVastrocytoma, glioblastoma, glioblastoma multiforme, small cell cancer,and non-small cell cancer, preferably non-small cell lung cancer, lungadenocarcinoma, metastatic melanoma, androgen-independent metastaticprostate cancer, androgen-dependent metastatic prostate cancer, prostateadenocarcinoma, and breast cancer, preferably breast ductal cancer,and/or breast carcinoma. In certain embodiments, the cancer treated withthe antibodies of this disclosure comprises glioblastoma. In certainembodiments, the cancer treated with one or more antibodies of thisdisclosure comprises pancreatic cancer. In certain embodiments, thecancer treated with one or more antibodies of this disclosure comprisesovarian cancer. In certain embodiments, the cancer treated with one ormore antibodies of this disclosure comprises lung cancer. In certainembodiments, the cancer treated with one or more antibodies of thisdisclosure comprises prostate cancer. In certain embodiments, the cancertreated with one or more antibodies of this disclosure comprises coloncancer. In certain embodiments, the cancer treated comprisesglioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostatecancer, or lung cancer. In a certain embodiment, the cancer isrefractory to other treatment. In a certain embodiment, the cancertreated is relapsed. In a certain embodiment, the cancer is arelapsed/refractory glioblastoma, pancreatic cancer, ovarian cancer,colon cancer, prostate cancer, or lung cancer.

In certain embodiments the cancer and or tumor to be treated with thecomposite binding molecules herein is a Mature B-cell neoplasm: ChronicLymphocytic Leukemia, Small Lymphocytic Lymphoma, Mantle Cell Lymphoma,Non-Hodgkins Lymphomas (Diffuse Large B-cell Lymphoma, FollicularLymphoma), Mucosa-associated lymphatic tissue (MALT) lymphoma,Mediastinal (thymic) large B-cell lymphoma, Lymphoplasmacytic lymphomaand Waldenstrom macroglobulinemia, Nodal marginal zone B-cell lymphoma,Splenic marginal zone lymphoma, Extranodal marginal zone B-celllymphoma, Intravascular large B-cell lymphoma, Primary effusionlymphoma, Burkitt lymphoma, or Primary central nervous system lymphoma.

In certain embodiments the cancer and or tumor to be treated with thecomposite binding molecules herein is a T cell neoplasm such as T-cellNon-Hodgkin Lymphoma, T-cell ALL, Mycosis Fungoides, Anaplastic LargeCell Lymphoma, Peripheral T-cell Lymphoma, T-Lymphocytic Leukemia(T-ALL), Acute Myeloblastic Leukemia, Acute Monocytic Leukemia, andothers.

In certain embodiments, the antibodies can be administered to a subjectin need thereof by any route suitable for the administration ofantibody-containing pharmaceutical compositions, such as, for example,subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral,or intracerebral, etc. In certain embodiments, the antibodies areadministered intravenously. In certain embodiments, the antibodies areadministered subcutaneously. In certain embodiments, the antibodies areadministered intratumoral. In certain embodiments, the antibodies areadministered on a suitable dosage schedule, for example, weekly, twiceweekly, monthly, twice monthly, once every two weeks, once every threeweeks, or once a month etc. In certain embodiments, the antibodies areadministered once every three weeks. The antibodies can be administeredin any therapeutically effective amount. In certain embodiments, thetherapeutically acceptable amount is between about 0.1 mg/kg and about50 mg/kg. In certain embodiments, the therapeutically acceptable amountis between about 1 mg/kg and about 40 mg/kg. In certain embodiments, thetherapeutically acceptable amount is between about 5 mg/kg and about 30mg/kg. Therapeutically effective amounts include amounts are thosesufficient to ameliorate one or more symptoms associated with thedisease or affliction to be treated.

Exemplary Embodiments

Provided herein are composite binding molecules comprising a CD19binding component configured to bind CD19 and a CD38 binding componentconfigured to bind CD38, wherein the CD19 binding component comprises anantibody or antigen binding fragment thereof and the CD38 bindingcomponent comprises an antibody or antigen binding fragment thereof. Insome embodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD19 and/or CD38 binding componentcomprise an immunoglobulin heavy and light chain pair, an scFv, a F(ab),a F(ab′)₂, a single domain antibody, a variable region fragment from animmunoglobulin new antigen receptor (VNAR), or a variable region derivedfrom a heavy chain antibody (VHH). In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe CD19 or CD38 binding component comprises an immunoglobulin heavy andlight chain pair. In some embodiments, provided is a composite bindingmolecule of any of the preceding embodiments, wherein the CD19 and CD38binding component comprise an immunoglobulin heavy and light chain pair.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the CD38 binding component comprisesan immunoglobulin heavy chain and an immunoglobulin light chain, whereinthe immunoglobulin heavy chain comprises an HCDR1 amino acid sequenceset forth in any one of SEQ ID NOs: 71-75, an HCDR2 amino acid sequenceset forth in any one of SEQ ID NOs: 81-85, or 150-155, an HCDR3 aminoacid sequence set forth in any one of SEQ ID NOs: 91-95; and theimmunoglobulin light chain comprises an LCDR1 amino acid sequence setforth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequenceset forth in any one of SEQ ID NOs: 111-115, and/or an LCDR3 amino acidsequence set forth in any one of SEQ ID NOs: 121-125; and wherein theCD19 binding component comprises an immunoglobulin heavy chain and animmunoglobulin light chain, wherein the immunoglobulin heavy chaincomprises an HCDR1 amino acid sequence set forth in any one of SEQ IDNOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ IDNOs: 21-25, an HCDR3 amino acid sequence set forth in any one of SEQ IDNOs: 31-35; and the immunoglobulin light chain comprises an LCDR1 aminoacid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and/oran LCDR3 amino acid sequence set forth in any one of SEQ ID NOs:121-125. In some embodiments, provided is a composite binding moleculeof any of the preceding embodiments, wherein the CD 38 binding componentcomprises an immunoglobulin heavy chain comprising an amino acidsequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:3; and the immunoglobulin light chain having at least about 90%, 95%,97%, 99% identity to SEQ ID NO: 4; and/or wherein the CD19 bindingcomponent comprises an immunoglobulin heavy chain comprising an aminoacid sequence having at least about 90%, 95%, 97%, 99% identity to SEQID NO: 1; and an immunoglobulin light chain having at least about 90%,95%, 97%, 99% identity to SEQ ID NO: 4. In some embodiments, provided isa composite binding molecule of any of the preceding embodiments,wherein the immunoglobulin heavy chain comprises an amino acid sequenceidentical to that set forth in SEQ ID NO: 3 or 5; and the immunoglobulinlight chain comprises an amino acid sequence identical to that set forthin SEQ ID NO: 4; and/or wherein the immunoglobulin heavy chain comprisesan amino acid sequence identical to that set forth in SEQ ID NO: 1 or 6;and the immunoglobulin light chain comprises an amino acid sequenceidentical to that set forth in SEQ ID NO: 4.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the composite binding molecule is acommon light chain bispecific IgG. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe CD 38 binding component comprises an immunoglobulin heavy chaincomprising an HCDR1 amino acid sequence set forth in any one of SEQ IDNOs: 71-75, an HCDR2 amino acid sequence set forth in any one of SEQ IDNOs: 81-85, or 150-155, an HCDR3 amino acid sequence set forth in anyone of SEQ ID NOs: 91-95; and the immunoglobulin light chain comprisesan LCDR1 amino acid sequence set forth in any one of SEQ ID NOs:101-105, an LCDR2 amino acid sequence set forth in any one of SEQ IDNOs: 111-115, and/or an LCDR3 amino acid sequence set forth in any oneof SEQ ID NOs: 121-125; and wherein the CD 19 binding componentcomprises an immunoglobulin heavy chain comprising an HCDR1 amino acidsequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acidsequence set forth in any one of SEQ ID NOs: 21-25, an HCDR3 amino acidsequence set forth in any one of SEQ ID NOs: 31-35; and theimmunoglobulin light chain comprises an LCDR1 amino acid sequence setforth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence setforth in any one of SEQ ID NOs: 51-55, and/or an LCDR3 amino acidsequence set forth in any one of SEQ ID NOs: 61-65. In some embodiments,provided is a composite binding molecule of any of the precedingembodiments, wherein the immunoglobulin heavy chain comprises an aminoacid sequence having at least about 90%, 95%, 97%, 99% identity to SEQID NO: 3 or 5; and the immunoglobulin light chain having at least about90%, 95%, 97%, 99% identity to SEQ ID NO: 4; and/or wherein theimmunoglobulin heavy chain comprises an amino acid sequence having atleast about 90%, 95%, 97%, 99% identity to SEQ ID NO: 1 or 7; and theimmunoglobulin light chain having at least about 90%, 95%, 97%, 99%identity to SEQ ID NO: 2.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the immunoglobulin heavy chaincomprises an amino acid sequence identical to that set forth in SEQ IDNO: 3 or 5; and the immunoglobulin light chain comprises an amino acidsequence identical to that set forth in SEQ ID NO: 4; and wherein theimmunoglobulin heavy chain comprises an amino acid sequence identical tothat set forth in SEQ ID NO: 1 or 7; and the immunoglobulin light chaincomprises an amino acid sequence identical to that set forth in SEQ IDNO: 2. In some embodiments, provided is a composite binding molecule ofany of the preceding embodiments, wherein the CD19 binding component orCD38 binding component comprise an scFv. In some embodiments, providedis a composite binding molecule of any of the preceding embodiments,wherein the CD19 binding component comprises an scFv. In someembodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD38 binding component comprises anscFv. In some embodiments, provided is a composite binding molecule ofany of the preceding embodiments, wherein the CD19 binding component orCD38 binding component comprise an immunoglobulin heavy-chain/lightchain pair. In some embodiments, provided is a composite bindingmolecule of any of the preceding embodiments, wherein the CD19 bindingcomponent comprises an immunoglobulin heavy-chain/light chain pair. Insome embodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD38 binding component comprises animmunoglobulin heavy-chain/light chain pair.

Further provided are composite binding molecules, wherein the compositebinding molecule comprises a CD38 antigen binding component that bindsCD38 comprising an anti-CD38 immunoglobulin heavy chain variable regionpaired with an anti-CD38 immunoglobulin light chain variable region anda CD19 antigen binding component that binds CD19 comprising an anti-CD19immunoglobulin heavy chain variable region paired with an anti-CD38immunoglobulin light chain variable region, wherein the CD38 antigenbinding component comprises: a) a heavy chain complementaritydetermining region 1 (HCDR1) comprising an amino acid sequence set forthin any one of SEQ ID NOs: 71-75; b) a heavy chain complementaritydetermining region 2 (HCDR2) comprising an amino acid sequence set forthin any one of SEQ ID NOs: 81-85, or 150-155, c) a heavy chaincomplementarity determining region 3 (HCDR3) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 91-95; d) a light chaincomplementarity determining region 1 (LCDR1) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 101-105; e) a light chaincomplementarity determining region 2 (LCDR2) comprising an amino acidsequence set forth in any one of SEQ ID NOs: 111-115; and/or f) a lightchain complementarity determining region 3 (LCDR3) comprising an aminoacid sequence set forth in any one of SEQ ID NOs: 121-125.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the CD19 antigen binding componentcomprises: g) a heavy chain complementarity determining region 1 (HCDR1)comprising an amino acid sequence set forth in any one of SEQ ID NOs:11-15, h) a heavy chain complementarity determining region 2 (HCDR2)comprising an amino acid sequence set forth in any one of SEQ ID NOs:21-25, i) a heavy chain complementarity determining region 3 (HCDR3)comprising an amino acid sequence set forth in any one of SEQ ID NOs:31-35; j) a light chain complementarity determining region 1 (LCDR1)comprising an amino acid sequence set forth in any one of SEQ ID NOs:101-105; k) a light chain complementarity determining region 2 (LCDR2)comprising an amino acid sequence set forth in any one of SEQ ID NOs:111-115; and/or 1) a light chain complementarity determining region 3(LCDR3) comprising an amino acid sequence set forth in any one of SEQ IDNOs: 121-125.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the CD38 antigen binding componentcomprises an immunoglobulin heavy chain variable region comprising anamino acid sequence having at least about 90%, 95%, 97%, 99% identity toSEQ ID NO: 3 or 5; and an immunoglobulin light chain variable regioncomprising an amino acid sequence having at least about 90%, 95%, 97%,99% identity to SEQ ID NO: 4. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe CD38 antigen binding component comprises an immunoglobulin heavychain variable region comprising an amino acid sequence identical to SEQID NO: 3 or 5; and an immunoglobulin light chain variable regioncomprises an amino acid sequence identical to SEQ ID NO: 4. In someembodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD19 antigen binding componentcomprises an anti-CD19 immunoglobulin heavy chain variable regioncomprising an amino acid sequence having at least about 90%, 95%, 97%,99% identity to SEQ ID NO: 1 or 6; and an immunoglobulin light chainvariable region comprising an amino acid sequence having at least about90%, 95%, 97%, 99% identity to SEQ ID NO: 4.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the anti-CD19 antigen bindingcomponent comprises an immunoglobulin heavy chain variable regioncomprising an amino acid sequence identical to SEQ ID NO: 1 or 6; and animmunoglobulin light chain variable region comprises an amino acidsequence identical to SEQ ID NO: 4. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe anti-CD38 immunoglobulin heavy chain variable region furthercomprises a first immunoglobulin heavy chain constant region. In someembodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the anti-CD38 immunoglobulin light chainvariable region further comprises an immunoglobulin light chain constantregion. In some embodiments, provided is a composite binding molecule ofany of the preceding embodiments, wherein the anti-CD19 immunoglobulinheavy chain variable region further comprises a second immunoglobulinheavy chain constant region. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe first immunoglobulin heavy chain constant region and/or the secondimmunoglobulin heavy chain constant region comprises one or more aminoacid substitutions that disfavors homodimerization of the anti-CD38immunoglobulin heavy chain constant region and/or promotesheterodimerization of the first heavy chain constant region and thesecond heavy chain constant region. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe one of the first or second immunoglobulin heavy chain constantregions comprises a T366W substitution (EU numbering), and the other ofthe first or second immunoglobulin heavy chain constant regionscomprises a T366S/L368A/Y407V substitution (EU numbering), such that theheterodimerization of the first and second immunoglobulin heavy chainconstant regions is favored compared to homodimerization of the first orsecond immunoglobulin heavy chain constant regions. In some embodiments,provided is a composite binding molecule of any of the precedingembodiments, wherein a single bispecific binding molecule is formed fromthe CD38 antigen binding component and the CD19 antigen bindingcomponent.

Also provided are composite binding molecules comprising a CD19 bindingcomponent that binds to CD19 and a CD38 binding component that binds toCD38, wherein the CD19 binding component comprises an scFV that binds toCD19, and the CD38 binding component comprises an immunoglobulinvariable region comprising a light-chain variable region and aheavy-chain variable region that bind to CD38. In some embodiments,provided is a composite binding molecule of any of the precedingembodiments, wherein the scFv that binds to CD19 is coupled to a firstimmunoglobulin heavy chain constant region. In some embodiments,provided is a composite binding molecule of any of the precedingembodiments, wherein the heavy-chain variable region of the CD38 bindingcomponent further comprises a second immunoglobulin heavy chain constantregion. In some embodiments, provided is a composite binding molecule ofany of the preceding embodiments, wherein the light-chain variableregion of the CD38 binding component further comprises an immunoglobulinlight chain constant region. In some embodiments, provided is acomposite binding molecule of any of the preceding embodiments, whereinthe CD19 binding component comprises an HCDR1 amino acid sequence setforth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence setforth in any one of SEQ ID NOs: 21-25, an HCDR3 amino acid sequence setforth in any one of SEQ ID NOs: 31-35; and the immunoglobulin lightchain comprises an LCDR1 amino acid sequence set forth in any one of SEQID NOs: 41-45, an LCDR2 amino acid sequence set forth in any one of SEQID NOs: 51-55, and/or an LCDR3 amino acid sequence set forth in any oneof SEQ ID NOs: 61-65.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the CD38 binding component comprisesan HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71-75,an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81-85,or 150-155, an HCDR3 amino acid sequence set forth in any one of SEQ IDNOs: 91-95; and the immunoglobulin light chain comprises an LCDR1 aminoacid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and/oran LCDR3 amino acid sequence set forth in any one of SEQ ID NOs:121-125. In some embodiments, provided is a composite binding moleculeof any of the preceding embodiments, wherein the CD19 binding componentcomprises an amino acid sequence having at least about 90%, 95%, 97%,99% identity to SEQ ID NO: 1 or 7; and the immunoglobulin light chainhaving at least about 90%, 95%, 97%, 99% identity to SEQ ID NO: 2. Insome embodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD38 binding component comprises anamino acid sequence having at least about 90%, 95%, 97%, 99% identity toSEQ ID NO: 3 or 5; and an amino acid sequence having at least about 90%,95%, 97%, 99% identity to SEQ ID NO: 4. In some embodiments, provided isa composite binding molecule of any of the preceding embodiments,wherein the CD19 binding component comprises an amino acid sequenceidentical to that set forth in SEQ ID NO: 1 or 7; and an amino acidsequence identical to that set forth in SEQ ID NO: 2. In someembodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD38 binding component comprises anamino acid sequence identical to that set forth in SEQ ID NO: 3 or 5;and an amino acid sequence identical to that set forth in SEQ ID NO: 4.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the first immunoglobulin heavy chainconstant region and/or the second immunoglobulin heavy chain constantregion comprises one or more amino acid substitutions that disfavorshomodimerization of the anti-CD38 immunoglobulin heavy chain constantregion and/or promotes heterodimerization of the first heavy chainconstant region and the second heavy chain constant region. In someembodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the one of the first or secondimmunoglobulin heavy chain constant regions comprises a T366Wsubstitution (EU numbering), and the other of the first or secondimmunoglobulin heavy chain constant regions comprises aT366S/L368A/Y407V substitution (EU numbering), such that theheterodimerization of the first and second immunoglobulin heavy chainconstant regions is favored over homodimerization of the first or secondimmunoglobulin heavy chain constant regions. In some embodiments,provided is a composite binding molecule of any of the precedingembodiments, wherein single bispecific binding molecule is formed fromthe CD38 antigen binding component and the CD19 antigen bindingcomponent.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein composite binding molecule is abispecific antibody or dual-antigen binding fragment thereof In someembodiments, provided is a composite binding molecule of any of thepreceding embodiments, comprising an Fc region comprising a nativecarbohydrate or an afucosylated carbohydrate modified amino acidresidue. In some embodiments, provided is a composite binding moleculeof any of the preceding embodiments, wherein the native carbohydrate orthe afucosylated carbohydrate modified amino acid residue corresponds toAsparagine 297 according to EU numbering.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the composite binding molecule bindsto CD19+, CD38+ B cells. In some embodiments, provided is a compositebinding molecule of any of the preceding embodiments, wherein thecomposite binding molecule binds exhibits reduced hemagglutinationcompared to a CD19 or CD38 monospecific antibody comprising an Fcregion.

Provided are nucleic comprising a polynucleotide sequence encoding thecomposite binding molecule of any one of composite binding molecules ofthe preceding embodiments. Embodiment 49: The nucleic acid of embodiment47, wherein the polynucleotide sequence encoding the composite bindingmolecule is operatively coupled to a eukaryotic regulatory sequence. Insome embodiments, provided is a cell comprising the nucleic acid ofembodiment any one of the preceding embodiments. In some embodiments,provided is a cell of any of the preceding embodiments, wherein the cellcomprises a prokaryotic cell. In some embodiments, provided is a cell ofany of the preceding embodiments, wherein the prokaryotic cell is anEscherichia coli cell. In some embodiments, provided is a cell of any ofthe preceding embodiments, wherein the cell comprises a eukaryotic cell.In some embodiments, provided is a cell of any of the precedingembodiments, wherein the eukaryotic cell is a Chinese Hamster Ovary(CHO) cell, an NSO murine myeloma cell, or a human PER.C6 cell.

Also provided are pharmaceutical compositions, for example, compositioncomprising the composite binding molecule of any one of the precedingembodiments and a pharmaceutically acceptable diluent, carrier, orexcipient. In some embodiments, composition is formulated forintravenous administration. In some embodiments, composition isformulated for subcutaneous administration.

Provided are composite binding molecules of any one of the precedingembodiments or the pharmaceutical composition of any one of thepreceding embodiments for use in a method of treating a tumor or acancer in an individual. In some embodiments, the tumor is a hematologiccancer. In some embodiments, the hematological cancer is a B cellmalignancy. In certain embodiments, the B cell malignancy is B-cellAcute Lymphocytic Leukemia. In certain embodiments, the B cellmalignancy is Chronic Lymphocytic Leukemia, Small Lymphocytic Lymphoma,Mantle Cell Lymphoma, or Non-Hodgkins Lymphomas (Diffuse Large B-cellLymphoma, Follicular Lymphoma). In some embodiments, the hematologicalcancer is a plasma malignancy. In certain embodiments, the plasmamalignancy is multiple myeloma. In some embodiments of any of thepreceding embodiments, the hematological cancer expresses CD19 and CD38(e.g. cells of the cancer express CD19 and CD38).

In some embodiments, the cancer or the tumor is a solid-tissue cancer.In some embodiments, the solid-tissue cancer comprises breast cancer,prostate cancer, pancreatic cancer, lung cancer, kidney cancer, stomachcancer, esophageal cancer, skin cancer, colorectal cancer, brain cancer,or head and neck cancer. In some embodiments, the breast cancer istriple negative breast cancer, the lung cancer is non-small cell lungcancer, the head and neck cancer is head and neck squamous cell cancer,the kidney cancer is renal cell carcinoma, the brain cancer isglioblastoma multiforme, or the skin cancer is melanoma.

Provided are also composite binding molecules of any one of thepreceding embodiments or the pharmaceutical composition of any one ofthe preceding embodiments for use in a method of reducingimmunosuppressive B cells in, adjacent to, or surrounding a tumor of anindividual. In some embodiments, Further provided are composite bindingmolecules of any one of the preceding embodiments or the pharmaceuticalcomposition of any one of the preceding embodiments for use in a methodof reducing immunosuppressive B cells in, adjacent to, or surrounding atumor of an individual. In some embodiments, the tumor infiltrating Bcells or the immunosuppressive B cells comprise CD19+, CD38+ B cells.

In some embodiments, provided is a composite binding molecule of any ofthe preceding embodiments, wherein the CD38 binding component comprisesa HCDR2 amino acid sequence comprising the sequence P-X1-LG-X2-A (SEQ IDNO: 156), wherein X1 and X2 are each selected from the group consistingof H, Q, T, N, S, G, A, R, K, D, or E. In certain embodiments, the X1 isH and X2 is T. In some embodiments, provided is a composite bindingmolecule of any of the preceding embodiments,X1 is H and X2 is T. Insome embodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein a heavy chain constant region of the CD19binding component comprises a A84S and/or A108L modification. In certainembodiments, provided is a composite binding molecule of any of thepreceding embodiments, wherein the CD38 binding component comprises alight chain sequence comprising a W32H substitution.

Further provided are methods of treating an individual afflicted with acancer or a tumor comprising administering to the individual afflictedwith the cancer or the tumor the composite binding molecule of any oneof the preceding embodiments or the pharmaceutical composition of anyone of the preceding embodiments, thereby treating the cancer or tumor.In some embodiments, the cancer or tumor is a hematologic cancer. Insome embodiments, the hematological cancer is a B cell malignancy. Incertain embodiments, the B cell malignancy is B-cell Acute LymphocyticLeukemia. In certain embodiments, the B cell malignancy is ChronicLymphocytic Leukemia, Small Lymphocytic Lymphoma, Mantle Cell Lymphoma,or Non-Hodgkins Lymphomas (Diffuse Large B-cell Lymphoma, FollicularLymphoma). In some embodiments, the hematological cancer is a plasmamalignancy. In certain embodiments, the plasma malignancy is multiplemyeloma. In some embodiments of any of the preceding embodiments, thehematological cancer expresses CD19 and CD38 (e.g. cells of the cancerexpress CD19 and CD38).

In some embodiments, the cancer or tumor is a solid-tissue cancer. Insome embodiments, the solid-tissue cancer comprises breast cancer,prostate cancer, pancreatic cancer, lung cancer, kidney cancer, stomachcancer, esophageal cancer, skin cancer, colorectal cancer, or head andneck cancer. In some embodiments, the breast cancer is triple negativebreast cancer, the lung cancer is non-small cell lung cancer, the headand neck cancer is head and neck squamous cell cancer, the kidney canceris renal cell carcinoma, the brain cancer is glioblastoma multiforme, orthe skin cancer is melanoma.

Provided are methods of reducing immunosuppressive B cells in, adjacentto, or surrounding a tumor of an individual afflicted with a tumor orcancer comprising administering to the individual afflicted with thetumor or the cancer the composite binding molecule of any one of thepreceding embodiments or the pharmaceutical composition of any one ofthe preceding embodiments, thereby reducing immunosuppressive B cells inthe tumor.

Also provided are methods of reducing immunosuppressive B cells in,adjacent to, or surrounding a tumor of an individual afflicted with atumor or cancer comprising administering to the individual afflictedwith the tumor or the cancer the composite binding molecule of any oneof the preceding embodiments or the pharmaceutical composition of anyone of the preceding embodiments, thereby reducing immunosuppressive Bcells in the tumor. In some embodiments, the tumor infiltrating B cellsor the immunosuppressive B cells comprise CD19+, CD38+ B cells.

Provided herein are also methods of making the composite bindingmolecule of any one of the preceding embodiments comprising incubatingthe cell of the preceding embodiments in a cell culture medium underconditions sufficient to allow expression, assembly, and secretion ofthe composite binding molecule into the cell culture medium. In someembodiments, the method comprises comprising isolating and purifying themolecule from the cell culture medium. Also provided are methods ofpreparing a cancer treatment for an individual comprising admixing thecomposite binding molecule of any one of the preceding embodiments witha pharmaceutically acceptable diluent, carrier, or excipient.

Thus, provided herein is a composite binding molecule comprising a firstbinding component configured to bind a first target and a second bindingcomponent configured to bind a second target, wherein the first targetcomprises a B-cell lineage surface marker, and wherein the second targetcomprises a suppressive B-cell surface marker, wherein the first targetand the second target are not identical. In some embodiments, the firstor the second binding component comprises a polypeptide. In certainembodiments, the first or the second binding component consists of apolypeptide. In some embodiments, the first and the second bindingcomponent comprise a polypeptide. In certain embodiments, the first andthe second binding component consist of a polypeptide. In someembodiments, the polypeptide of the first or second binding componentcomprises an amino acid sequence at least 100 amino acid residues inlength. In some embodiments, the polypeptide of the first and secondbinding component comprise an amino acid sequence at least 100 aminoacid residues in length.

The B-cell lineage surface marker can comprise CD19, CD138, IgA, orCD45. In some embodiments, the B-cell lineage surface marker comprisesCD19. In certain embodiments, the B-cell lineage surface marker is CD19.In some embodiments, the B-cell lineage surface marker is IgA. Incertain embodiments, the B-cell lineage surface marker is IgA. In someembodiments, the B-cell lineage surface marker is CD138. In certainembodiments, the B-cell lineage surface marker is CD138. In someembodiments, the B-cell lineage surface marker is CD45. In certainembodiments, the B-cell lineage surface marker is CD45. In someembodiments, the B-cell lineage surface marker is selected from thegroup consisting of IgA, CD19, CD138, CD45, and any combination thereof

The suppressive B-cell surface marker can comprise IgD, CD1, CD5, CD21,CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-beta (e.g., TGF-beta LAP).In some embodiments, the suppressive B-cell surface marker comprisesIgD. In certain embodiments, the suppressive B-cell surface marker isIgD. In some embodiments, the suppressive B-cell surface markercomprises CD1. In certain embodiments, the suppressive B-cell surfacemarker is CD1. In some embodiments, the suppressive B-cell surfacemarker comprises CD5. In certain embodiments, the suppressive B-cellsurface marker is CD5. In some embodiments, the suppressive B-cellsurface marker comprises CD21. In certain embodiments, the suppressiveB-cell surface marker is CD21. In some embodiments, the suppressiveB-cell surface marker comprises CD24. In certain embodiments, thesuppressive B-cell surface marker is CD24. In some embodiments, thesuppressive B-cell surface marker comprises CD38. In certainembodiments, the suppressive B-cell surface marker is CD38. In someembodiments, the B-cell surface marker is selected from the groupconsisting of IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3,latent TGF-beta (e.g., TGF-beta LAP), and any combination thereof.

The composite binding molecule can comprise an antibody ortarget-binding fragments thereof. In some embodiments, the first orsecond binding component comprise an immunoglobulin heavy and lightchain pair, an scFv, a F(ab), a F(ab′)₂, a single domain antibody, avariable region fragment from an immunoglobulin new antigen receptor(VNAR), or a variable region derived from a heavy chain antibody (VHH).In some embodiments, the first and second binding component comprise animmunoglobulin heavy and light chain pair, an scFv, a F(ab), a F(ab′)₂,a single domain antibody, a variable region fragment from animmunoglobulin new antigen receptor (VNAR), or a variable region derivedfrom a heavy chain antibody (VHH). In certain embodiments, the first orsecond binding component comprises an immunoglobulin heavy and lightchain pair. In certain embodiments, the first and second bindingcomponent comprise an immunoglobulin heavy and light chain pair. Incertain embodiments, the first or second binding component comprises anscFv. In certain embodiments, the first and second binding componentcomprise an scFv.

The composite binding molecule described herein, wherein compositebinding molecule is a bispecific antibody or dual-antigen bindingfragment thereof

In some embodiments, the composite binding molecule comprises animmunoglobulin heavy chain and an immunoglobulin light chain, whereinthe immunoglobulin heavy chain comprises an HCDR1 amino acid sequenceset forth in any one of SEQ ID NOs: 71-75, an HCDR2 amino acid sequenceset forth in any one of SEQ ID NOs: 81-85, or 150-155, an HCDR3 aminoacid sequence set forth in any one of SEQ ID NOs: 91-95; and animmunoglobulin light chain comprises an LCDR1 amino acid sequence setforth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence setforth in any one of SEQ ID NOs: 51-55, and/or an LCDR3 amino acidsequence set forth in any one of SEQ ID NOs: 61-65. In certainembodiments, the immunoglobulin heavy chain comprises an amino acidsequence having at least about 90%, 95%, 97%, 99% identity to SEQ ID NO:3; and the immunoglobulin light chain having at least about 90%, 95%,97%, 99% identity to SEQ ID NO: 2. In certain embodiments, theimmunoglobulin heavy chain comprises an amino acid sequence identical tothat set forth in SEQ ID NO: 3; and the immunoglobulin light chaincomprises an amino acid sequence identical to that set forth in SEQ IDNO: 2. In some embodiments, the composite binding molecule is a commonlight chain bispecific IgG.

The composite binding molecule can be a bispecific antibody. In someembodiments, the bispecific antibody is selected from one of thefollowing formats: a common light chain bispecific IgG, a Fab-Fc:scFv-Fcbispecific IgG, a Fab-Fc-Fab:Fc bispecific IgG, aFab-Fc-scFv:Fab-Fc-scFv bispecific IgG, a Fab-Fc-scFv:Fc bispecific IgG,a Fab-Fc-Fab:Fab-Fc bispecific IgG, an scFv-Fab-Fc:scFv-Fab-Fcbispecific IgG, a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG, aFab-Fc-Fab:Fab-Fc-Fab bispecific IgG, and a Fab-Fc-scFv:Fab-Fcbispecific IgG. In certain embodiments, the bispecific antibody is acommon light chain bispecific IgG. In certain embodiments, thebispecific antibody is a Fab-Fc:scFv-Fc bispecific IgG. In certainembodiments, the bispecific antibody is a Fab-Fc-Fab:Fc bispecific IgG.In certain embodiments, the bispecific antibody is aFab-Fc-scFv:Fab-Fc-scFv bispecific IgG. In certain embodiments, thebispecific antibody is a Fab-Fc-scFv:Fc bispecific IgG. In certainembodiments, the bispecific antibody is a Fab-Fc-Fab:Fab-Fc bispecificIgG. In certain embodiments, the bispecific antibody is anscFv-Fab-Fc:scFv-Fab-Fc bispecific IgG. In certain embodiments, thebispecific antibody is a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG. Incertain embodiments, the bispecific antibody is a Fab-Fc-Fab:Fab-Fc-Fabbispecific IgG. In certain embodiments, the bispecific antibody is anIgG-scFv

The composite binding molecule can comprise post-translationalmodification. In some embodiments, the composite binding moleculecomprises an Fc region comprising a native carbohydrate or anafucosylated carbohydrate modified amino acid residue. In certainembodiments, the native carbohydrate or the afucosylated carbohydratemodified amino acid residue corresponds to Asparagine 297 according toEU numbering.

In some embodiments, the first binding component comprises an HCDR1amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2amino acid sequence set forth in any one of SEQ ID NOs: 21-25, an HCDR3amino acid sequence set forth in any one of SEQ ID NOs: 31-35, an LCDR1amino acid sequence set forth in any one of SEQ ID NOs: 41-45, an LCDR2amino acid sequence set forth in any one of SEQ ID NOs: 51-55, and/or anLCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61-65. Incertain embodiments, the first binding component comprises an amino acidsequence comprising at least about 90%, 95%, 97%, 99% identity to, or is100% identical to the amino acid sequence set forth in any one of SEQ IDNOs: SEQ ID NO: 1 and SEQ ID NO: 2. In certain embodiments, the firstbinding component comprises an amino acid sequence comprising at leastabout 90%, 95%, 97%, 99% identity to, or is 100% identical to the aminoacid sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 2.

In some embodiments, the second binding component comprises an HCDR1amino acid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or150-155, an HCDR3 amino acid sequence set forth in any one of SEQ IDNOs: 91-95, an LCDR1 amino acid sequence set forth in any one of SEQ IDNOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQID NOs: 111-115, and/or an LCDR3 amino acid sequence set forth in anyone of SEQ ID NOs: 121-125. In certain embodiments, the second bindingcomponent comprises an amino acid sequence comprising at least about90%, 95%, 97%, 99% identity to, or is 100% identical to the amino acidsequence set forth in any one of SEQ ID NO: 3 and SEQ ID NO: 4. Incertain embodiments, the second binding component comprises an aminoacid sequence comprising at least about 90%, 95%, 97%, 99% identity to,or is 100% identical to the amino acid sequence set forth in SEQ ID NO:SEQ ID NO: 3 and SEQ ID NO: 4.

The composite binding molecule can bind a first target and a secondtarget, wherein the first target comprises a B-cell lineage surfacemarker, and wherein the second target comprises a suppressive B-cellsurface marker. In some embodiments, the composite binding moleculebinds to CD19 positive (CD19+ or CD19^(high)) and CD38 positive (CD38+or CD19^(high)) B-cells.

The composite binding molecule can be encoded for by a nucleic acidmolecule. Disclosed herein are nucleic acids comprising a polynucleotidesequence encoding a composite binding molecule disclosed herein. In someembodiments, the polynucleotide sequence encoding the composite bindingmolecule is operatively coupled to a eukaryotic regulatory sequence.

A cell can comprise the nucleic acid encoding the composite bindingmolecules. In some embodiments, the cell comprises a prokaryotic cell.In certain embodiments, the prokaryotic cell is an Escherichia colicell. In come embodiments, the cell comprises a eukaryotic cell. Incertain embodiments, the eukaryotic cell is a Chines Hamster Ovary (CHO)cell, an NS0 murine myeloma cell, or a human PER.C6 cell.

Also disclosed herein are compositions comprising the composite bindingmolecule and a pharmaceutically acceptable diluent, carrier, orexcipient. In some embodiments, the compositions are formulated forintravenous administration. In some embodiments, the compositions areformulated for subcutaneous administration.

The composite binding molecule disclosed herein can inhibit and/orreduce the number of immunosuppressive B cells that suppress ananti-tumor immune response. Thus, the composite binding molecules hereincan be used in a method of treating a tumor or a cancer in anindividual. In some embodiments, the cancer or the tumor is ahematologic cancer. In some embodiments, the hematological cancer is a Bcell malignancy. In certain embodiments, the B cell malignancy is B-cellAcute Lymphocytic Leukemia. In certain embodiments, the B cellmalignancy is Chronic Lymphocytic Leukemia, Small Lymphocytic Lymphoma,Mantle Cell Lymphoma, or Non-Hodgkins Lymphomas (Diffuse Large B-cellLymphoma, Follicular Lymphoma). In some embodiments, the hematologicalcancer is a plasma malignancy. In certain embodiments, the plasmamalignancy is multiple myeloma. In some embodiments of any of thepreceding embodiments, the hematological cancer expresses CD19 and CD38(e.g. cells of the cancer express CD19 and CD38).

In some embodiments, the cancer or the tumor is a solid-tissue cancer.In some embodiments, the cancer comprises breast cancer, prostatecancer, pancreatic cancer, lung cancer, kidney cancer, stomach cancer,esophageal cancer, skin cancer, colorectal cancer, or head and neckcancer. In some embodiments, the cancer is breast cancer. In somecertain embodiments, the breast cancer is triple negative breast cancer.In some embodiments, the cancer is lung cancer. In certain embodiments,the lung cancer is non-small cell lung cancer. In some embodiments, thecancer is head and neck cancer. In certain embodiments, the head andneck cancer is head and neck squamous cell cancer. In some embodiments,the cancer is kidney cancer. In certain embodiments, the kidney canceris renal cell carcinoma. In some embodiments, the cancer is braincancer. In some embodiments, the brain cancer is glioblastomamultiforme. In some embodiments, the cancer is skin cancer. In certainembodiments, the skin cancer is melanoma.

The composite binding molecules herein can be used in a method ofreducing tumor infiltrating B cells and/or immunosuppressive B cellsthat suppress an anti-tumor immune response against a tumor of anindividual. The composite binding molecules herein can be used in amethod of inhibiting the function of tumor infiltrating B cells and/orimmunosuppressive B cells that suppress an anti-tumor immune responseagainst a tumor of an individual. The composite binding molecule can beused in a method of reducing suppressive B cells in, adjacent to, orsurrounding a tumor of an individual. In some embodiments, the tumorinfiltrating B cells or the immunosuppressive B cells comprise CD19+,CD38+ B cells.

Further disclosed herein are methods of treating an individual afflictedwith a cancer or a tumor comprising administering to the individualafflicted with the cancer or the tumor the composite binding moleculedisclosed herein, thereby treating the cancer or tumor. In someembodiments, the cancer or tumor is a hematologic cancer. In someembodiments, the hematological cancer is a B cell malignancy. In certainembodiments, the B cell malignancy is B-cell Acute Lymphocytic Leukemia.In certain embodiments, the B cell malignancy is Chronic LymphocyticLeukemia, Small Lymphocytic Lymphoma, Mantle Cell Lymphoma, orNon-Hodgkins Lymphomas (Diffuse Large B-cell Lymphoma, FollicularLymphoma). In some embodiments, the hematological cancer is a plasmamalignancy. In certain embodiments, the plasma malignancy is multiplemyeloma. In some embodiments of any of the preceding embodiments, thehematological cancer expresses CD19 and CD38 (e.g. cells of the cancerexpress CD19 and CD38).

In some embodiments, the cancer or tumor is a solid-tissue cancer. Insome embodiments, the cancer comprises breast cancer, prostate cancer,pancreatic cancer, lung cancer, kidney cancer, stomach cancer,esophageal cancer, skin cancer, colorectal cancer, or head and neckcancer. In some embodiments, the cancer is breast cancer. In somecertain embodiments, the breast cancer is triple negative breast cancer.In some embodiments, the cancer is lung cancer. In certain embodiments,the lung cancer is non-small cell lung cancer. In some embodiments, thecancer is head and neck cancer. In certain embodiments, the head andneck cancer is head and neck squamous cell cancer. In some embodiments,the cancer is kidney cancer. In certain embodiments, the kidney canceris renal cell carcinoma. In some embodiments, the cancer is braincancer. In some embodiments, the brain cancer is glioblastomamultiforme. In some embodiments, the cancer is skin cancer. In certainembodiments, the skin cancer is melanoma.

Also disclosed is a method of reducing tumor infiltrating B cells in,adjacent to, or surrounding a tumor of an individual afflicted with atumor or cancer comprising administering to the individual afflictedwith the tumor or the cancer the composite binding molecule disclosedherein, thereby reducing tumor infiltrating B cells in the tumor. Alsodisclosed are methods of reducing immunosuppressive B cells in, adjacentto, or surrounding a tumor of an individual afflicted with a tumor orcancer comprising administering to the individual afflicted with thetumor or the cancer the composite binding molecule disclosed herein,thereby reducing immunosuppressive B cells in the tumor. In someembodiments, the tumor infiltrating B cells or the immunosuppressive Bcells comprise CD19+, CD38+ B cells. In some embodiments, reducing tumorinfiltrating B cells comprises reducing and/or blocking and/orpreventing and/or inhibiting the recruitment of immunosuppressive Bcells into a tumor environment or microenvironment. In some embodiments,reducing tumor infiltrating B cells comprises reducing and/or blockingand/or preventing and/or inhibiting cell-to-cell contact inducedimmunosuppression mediated by immunosuppressive B cells. In someembodiments, reducing tumor infiltrating B cells comprises reducingand/or blocking and/or preventing and/or inhibiting immunosuppressive Bcell differentiation.

Disclosed herein are method of making the composite binding moleculedisclosed herein, comprising incubating the cell disclosed herein in acell culture medium under conditions sufficient to allow expression,assembly and secretion of the composite binding molecule into the cellculture medium. In some embodiments, the method comprised isolating andpurifying the molecule from the cell culture medium.

The composite binding molecule disclosed herein can be used in treatinga cancer or tumor. Thus, disclosed is a method of preparing a cancertreatment for an individual comprising admixing a composite bindingmolecule of the disclosure with a pharmaceutically acceptable diluent,carrier, or excipient.

EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Cell Binding Properties of CD19 and CD38 Antibodies

Exemplifying the disclosure herein, composite binding moleculecomprising a first binding component configured to bind a first targetand a second binding component configured to bind a second target,wherein the first target comprises a B-cell lineage surface marker, andwherein the second target comprises a suppressive B-cell surface marker,were tested for binding to cells expressing CD19 and CD38. The bindingproperties of antibodies comprising CD19 and CD38 light and heavy chainsto Raji cells expressing CD19 and CD38. FIG. 12A shows cell surfaceexpression data of CD19 and CD38 in Raji cells. Raji cells expressingCD19 and CD38 were incubated with antibodies comprising CD19 and CD38light and heavy chains. Cells were incubated with 30 ug/mL of antibodiesacross 11 differing concentrations to generate a binding profile foreach sample. expression of CD19 and CD38 was validated usingcommercially available antibodies. Samples tested include: (A) matchedCD19 heavy and light chains, the CD19 heavy chain comprising SEQ ID NO:1 and CD19 light chain comprising SEQ ID NO: 2; (B) swapped CD19 heavyand CD38 light chains, the CD19 heavy chain comprising SEQ ID NO: 1 andCD38 light chain comprising SEQ ID NO: 4; (C) swapped CD38 heavy andCD19 light chains, the CD38 heavy chain comprising SEQ ID NO: 3 and CD19light chain comprising SEQ ID NO: 2; (D) matched CD38 heavy and lightchains, the CD38 heavy chain comprising SEQ ID NO: 3 and CD38 lightchain comprising SEQ ID NO: 4; (E) a CD19 single chain variable fragment(scFv) comprising SEQ ID NO:1-2; (F) a CD38 single chain variablefragment (scFv) comprising SEQ ID NO:3-4; Darzalex® (CD38 control);Anti-CD19 PE (CD19 control); Anti-CD38 PE (CD38 control); and an IgG1isotype control

FIG. 12B and 12C show binding profiles of samples A-F, Darzalex®, andthe IgG1 isotype control. TABLE 1 and TABLE 2 shows ECso values andmaximum mean fluorescence intensity (MFI) of samples A-F, Darzalex®, andthe IgG1 isotype control. Each of samples A-F demonstrated binding toRaji cells expressing CD19 and CD38, wherein the binding profiles ofsamples A-F varied amongst the samples. FIG. 12D and 12E show binding ofcontrol anti-CD19 (FIG. 11D) and anti-CD38 antibodies (FIG. 11E). FIG.12F shows that the antibodies tested did not bind to CHO cells that donot express CD19 and CD38.

TABLE 1 Cell Binding Sample EC50 (ng/mL) Max MFI A ~6E+21 1878626 B~38394212 1842964 C ND ND D 2260 3827997 E ~1E+16 3957921 F 378894416606 Darzalex ® 195 4016941 IgG1 isotype control ~4622880 38691

TABLE 2 Cell Binding Sample EC50 (nM) Max MFI Adjusted* EC50 (nM) A NA1878626 78.79 B NA 1842964 113.11 C ND ND ND D 17.39 3827997 17.39 E NA3957921 66.85 F NA 4416606 67.40 Darzalex ® 1.27 4016941 1.27 IgG1isotype control — 38691 — NA The treatment did not produce a signal thatplateaued. EC50 cannot be defined in the conventional manner *The topasymptote is fixed at about the maximum response of Darzalex ®.

Example 2 Octet® Binding Data

The binding affinities of parental and bispecific antibodies weredetermined using bio-layer interferometry. Binding experiments wereperformed on Octet® Red96 at 25° C. using an assay Buffer consisting of0.1% BSA, 1XPBS, 0.02% Tween®-20, 0.05% NaN3. The antibodies were loadedonto Anti-hIgG Fc Capture biosensors for 300 seconds. The ligand-loadedsensors were dipped into a series dilution (starting at 300 nM: two-foldseries dilution for CD19 and three-fold series dilution for CD38) of theantigens for association (200 seconds for CD19 and 150 seconds for CD38)followed by dissociation (600 seconds for CD19 and 400 seconds forCD38). Kinetic constants were calculated using a monovalent (1:1)binding model.

Parental test articles included:

851A=anti-CD19 3C10

851B=anti-CD19 3C10 heavy chain & anti-CD38 003 light chain

851C=anti-CD38 003 heavy chain & anti-CD19 3C10 light chain

851D=anti-CD38 003

851E=anti-CD19 3C10 (scFv-Fc)2

851F=anti-CD38 003 (scFv-Fc)2

The two parental antibodies with anti-CD19 3C10 VH and VL (851A/851E)bound CD19 with a similar KD. Substituting the anti-CD19 3C10 VL withthe anti-CD38 VL (851B) resulted in a reduction of binding to CD19 ofabout 5-fold. The parental antibodies with anti-CD38 003 VH and VL(851D/851F) did not bind to CD19, as expected.

Table 3 shows binding data. The two parental antibodies with anti-CD38003 VH and VL (851D/851F) bound CD38 with a similar KD. Substituting theanti-CD38 003 VL with the anti-CD19 VL (851C) resulted in a largereduction of binding to CD38. The parental antibodies with anti-CD19 VHand VL (851A/851E) did not bind to CD38, as expected; 851B also did notbind to CD38. This data shows that only the anti-CD38 003 VL canfunction as a common light chain for the anti-CD19 3C10 VH.

TABLE 3 KD (nM) Sample CD19 CD38 851A 1.53 NB 851B 7.07 NB 851C NB 285851D NB 0.98 851E 1.21 NB 851F NB 2.22 NB = no binding

Bispecific antibody (format) test articles included:

BS1=1:1:2 ratio 003HC:3C10HC:003LC (common light chain)

BS1b=2:1:2 ratio 003HC:3C10HC:003LC (common light chain)

BS2=1:1:1 ratio 003Knob:3C10scFvHole:003LC (Fab-Fc:scFv-Fc bispecificIgG1)

BS2b=4:1:4 ratio 003Knob:3C10scFvHole:003LC (Fab-Fc:scFv-Fc bispecificIgG1)

BS3=1:1:1 ratio 3C10scFv-003Fab-FcKnob:FcHole:003LC) (scFv-Fab-Fc:Fcbispecific IgG1)

BS4=1:1:1 ratio 003Fab-FcKnob-3C10scFv:FcHole (Fab-Fc-scFv:Fc bispecificIgG1)

BS4b=4:1:4 ratio 003Fab-FcKnob-3C10scFv:FcHole (Fab-Fc-scFv:Fcbispecific IgG1)

CM1=1:1:2 ratio 3C10Hole:VZVKnob:003LC anti-CD19 control antibody

CM1b=1:3:3 ratio 3C10Hole:VZVKnob:003LC

CM2=1:1:2 ratio 003Knob:VZVHole:003LC anti-CD38 control antibody

CM2b=3:1:3 ratio 003Knob:VZVHole:003LC

Table 4 shows binding data for bispecific test articles in a singleantigen format. Bispecific antibodies BS1/BS2/BS4 bound to both targetantigens with a KD within 4-fold of parental antibodies (shown with grayshading). BS3 bound only to CD19 but not CD38 suggesting that either theanti-CD38 Fab binding site was blocked by the anti-CD19 scFv N-terminalfusion or the anti-CD38 requires a free VH N-terminus for binding.One-arm control antibodies (CM1, CM2) bound only to their intendedtarget antigen.

TABLE 4 KD (nM) Sample CD19 CD38 BS1 13.8 1.32 BS1b 13.0 1.23 BS2 1.531.33 BS2b 1.58 1.12 BS3 3.32 NB BS4 1.31 1.20 BS4b 4.78 1.21 CM1 18.2 NBCM1b 15.9 2210 CM2 NB 1.67 CM2b 21300 0.59 NB = no binding

For a two-antigen format, the antibodies were loaded onto Anti-hlgG FcCapture biosensors for 300 seconds. The ligand-loaded sensors weresaturated with 500 nM of first antigen for 500 seconds followed by 300nM of second antigen for 240 seconds. Kinetic constants were calculatedusing a monovalent (1:1) binding model. Table 5 shows bispecificantibodies BS1/BS2/BS4 could simultaneously bind to both target antigenswith a ka (1/Ms) within 2-fold of parental antibodies (851B, 851D, and851E). As with the one-antigen format, BS3 bound only to CD19 but notCD38.

TABLE 5 Second KD (nM) Antigen Ka Sample Antigen 1 Antigen 2 (1/Ms) E+04851B — CD19 4.24 851D — CD38 43.1 851E — CD19 4.89 BS1 CD19 CD38 27.0BS1 Buffer CD38 40.9 BS1b CD19 CD38 26.3 BS1b Buffer CD38 31.6 BS2 CD19CD38 25.4 BS2 Buffer CD38 44.7 BS2b CD19 CD38 22 BS2b Buffer CD38 32.8BS3 CD19 CD38 NB BS3 Buffer CD38 NB BS4 CD19 CD38 37.2 BS4 Buffer CD3844.5 BS4b CD19 CD38 26.7 BS4b Buffer CD38 39.0 CM1 CD19 CD38 NB CM1Buffer CD38 NB CM2 CD19 CD38 30.0 CM2 Buffer CD38 29.4 BS1 CD38 CD194.44 BS1 Buffer CD19 5.34 BS1b CD38 CD19 6.87 BS1b Buffer CD19 8.98 BS2CD38 CD19 5.74 BS2 Buffer CD19 5.31 BS2b CD38 CD19 6.66 BS2b Buffer CD198.20 BS3 CD38 CD19 8.84 BS3 Buffer CD19 8.93 BS4 CD38 CD19 2.65 BS4Buffer CD19 4.55 BS4b CD38 CD19 3.29 BS4b Buffer CD19 4.64 CM1 CD38 CD195.07 CM1 Buffer CD19 4.73 CM2 CD38 CD19 NB CM2 Buffer CD19 NB NB = nobinding

Variants were further tested for the ability to bind CD19 and/or CD38.Binding experiments were performed on Octet® Red at 25° C. Theantibodies were loaded onto anti-hIgG Fc Capture (AHC) biosensors for300 seconds. The ligand-loaded sensors were dipped into a two-foldseries dilution (starting at 300 nM) of the antigens (CD19 and CD38) for240 seconds of CD19 and 150 seconds of CD38 for association followed bydissociation for 600 seconds of CD19 and 130 seconds of CD38. Kineticconstants were calculated using a monovalent (1:1) binding model. TABLE6 shows binding of anti-CD38 CDRH2 variants. TABLE 7 shows binding ofthe CD38 light chain W32H variant. TABLE 8 shows binding of CD19 heavychain framework mutant A84S A108L.

TABLE 6  Bispecific BS1 anti-CD38  arm CDR-H2 variants (“RVIPFLGIAN” disclosed as SEQ ID NO: 85) CD38  Binding SEQUENCE KD (nM) BS1 R V I P FL G I A N 1.2 BS1M-1 . . . . . . . T . . 13.2 BS1M-3 . . . . H . . . .6.9 BS1M-4 . . . . H . . T . . 94.4 BS1M-6 . . . . Q . . . . 4.4 BS1M-7. . . . Q . . T . . 60.5 BS1M-2 . . T . . . . T . . No binding BS1M-5 ..  T . H . . T .  . No binding BS1M-8 . .  . . H Q . T .  . No bindingBS1M-9 . .  . . Q Q . T .  . No binding

TABLE 7 Bispecific BS1 common light chain variant CD38 Binding CD19Binding KD (nM) KD (nM) BSM-10 (W32H) 77.3 39.4 BS1 1.2 13

TABLE 8 Bispecific BS1 anti-CD19 arm framework variant CD19 Binding KD(nM) BSM-14 (A84S A108L) 9.5 BS1 13

Example 3 Cell Binding Studies

Cell Binding Studies Protocol: Five cell lines (HEK293-CD19,HEK293-CD38, HEK293-CD19/CD38, Daudi, and REH) were incubated with testarticles at 133 nM followed by a 3-fold dilution series (7 pointstotal), in addition to a no treatment control, in triplicate. The HEK293cell lines were transiently transfected.

A study was performed to evaluate the cell surface expression of CD19and CD38 on Daudi, Raji and REH cell lines. Cells were stained, intriplicate, with commercially available antibody conjugated to PE,washed, and acquired via flow cytometry. To quantify the moleculeexpression on the surface of the cells, a Quantum Simply Cellularanti-mouse IgG kit from Bangs Laboratories (Catalog #815-A) was used togenerate a standard curve for interpolating MFI to a molecule number percell value (Table 9).

TABLE 9 Number of Molecules Cell Line CD19 CD38 Daudi 200,000 1,000,000Raji 200,000 1,000,000 REH 50,000 300,000

FIG. 13A shows binding to Daudi cells of the parental antibodies (851A,851B, 851D) and the two control bispecific antibodies (each with one armagainst CD19 or CD38 and the other arm against varicella zoster virus).Given that the Daudi cells have ˜1 million copies of CD38 on theirsurface but only ˜200,000 copies of CD19, FIG. 13A shows efficientbinding of anti-CD38 851D and 38K-VZVH but only moderate binding of theanti-CD19 851A, 851B, 19H-VZVK. Note that 851D with two CD38 bindingFabs binds about 5-fold better than 38K-VZVH, which has only one bindingFab for CD38.

FIG. 13B shows binding to Daudi cells of bispecific antibodies BS1, BS2and BS4. The avidity of the bispecific antibodies, binding to both CD38and CD19, is apparent by comparing their binding to the 38K-VZVH, whichbinds only to CD38.

FIG. 14A shows binding to REH cells of the parental antibodies (851A,851B, 851D) and the two control bispecific antibodies (each with one armagainst CD19 or CD38 and the other arm against varicella zoster virus).Given that the REH cells have ˜300,000 copies of CD38 on their surfacebut only ˜50,000 copies of CD19, FIG. 14A shows efficient binding ofanti-CD38 851D and 38K-VZVH but only moderate binding of the anti-CD19851A, 851B, 19H-VZVK. The magnitude of MFI is significantly lesscompared to Daudi cells (FIGS. 2A, 2B) due to the lower expression levelof both CD38 and CD19 on REH cells. Note that 851D with two CD38 bindingFabs binds about 5-fold better than 38K-VZVH, which has only one bindingFab for CD38.

FIG. 14B shows binding to REH cells of bispecific antibodies BS1, BS2and BS4. The avidity of the bispecific antibodies, binding to both CD38and CD19, is apparent by comparing their binding to the 38K-VZVH, whichbinds only to CD38.

FIG. 15A shows binding to CD19-transfected HEK293 cells of the parentalantibodies (851A, 851B, 851D) and two control bispecific antibodies(38K-VZVH, 19H-VZVK). As expected, the two anti-CD38 antibodies do notbind to these cells. Note that 851A and 851B, each with two CD19 bindingFabs, bind significantly better than 19H-VZVK, which has only onebinding Fab for CD19.

FIG. 15B shows binding to CD19-transfected HEK293 cells of bispecificantibodies BS1, BS2 and BS4. BS2 and BS$ bind slightly better than BS1;BS2 and BS4 bind CD19 about 10-fold better than BS1 since BS1 has theanti-CD38 light chain (see Table Octet® data).

FIG. 16A shows binding to CD38-transfected HEK293 cells of the parentalantibodies (851A, 851B, 851D) and two control bispecific antibodies(38K-VZVH, 19H-VZVK). As expected, the three anti-CD19 antibodies do notbind to these cells. Note that 851D, with two CD38 binding Fabs, bindsbetter than 38K-VZVH, which has only one binding Fab for CD38.

FIG. 16B shows binding to CD38-transfected HEK293 cells of bispecificantibodies BS1, BS2 and BS4.

Cell Binding Studies Protocol - Non-Specific Background Binding: A studywas performed to evaluate the binding of three parental monoclonalantibodies (anti-CD19 clones 851A and 851B and anti-CD38 clone 851D), ahuman IgG1 isotype control, and daratumumab to CHO-S and Expi293T celllines. The two cell lines were stained with a viability dye, thenincubated with test articles at a top concentration of 1,250 nM followedby a 5-fold dilution series (4 points total), in addition to a notreatment control, as well as a no treatment, no secondary control, intriplicate.

FIG. 17A shows binding to non-transfected CHO-S cells of the parentalantibodies (851A, 851B, 851D). Non-specific binding was seen beginningat 250nM for all three parental antibodies and was more pronounced foranti-CD38 851D.

FIG. 17B shows binding to non-transfected Expi293T cells of the parentalantibodies (851A, 851B, 851D). Non-specific binding was seen beginningat 250nM for all three parental antibodies and was more pronounced foranti-CD38 851D.

Example 4 Direct and Cross-Linked Apoptosis

For assessment of direct apoptosis, cells were treated with testarticles and incubated for 48 hours at 37° C/5% CO2. For assessment ofcross-linking induced apoptosis, cells were incubated with test articleson ice for 30 minutes prior to the addition of rabbit anti-human Fcgamma specific F(ab′)2 at 5μg/mL. Cells were then incubated for 48 hoursat 37C/5% CO2. After incubation, cells were washed and stained withAnnexin V, then resuspended in Annexin V buffer containing a viabilitydye (propidium iodide; PI) prior to flow cytometry acquisition. Earlyapoptotic cells were defined as Annexin V+/PI− single cells, while lateapoptotic/necrotic cells were defined as Annexin V+/PI+ single cells.The sum of Annexin V+/PI− and Annexin V+/PI− were defined as totalapoptotic/necrotic cells. The percentages of Annexin V+/PI− cells orAnnexin V+/PI+ were plotted to compare the various apoptosis conditions.

For direct apoptosis assessment, test articles were each tested at afinal top concentration of 33 nM, followed by a 7-point five-folddilution series, in addition to an untreated control, in triplicate. Forcross-linking induced apoptosis, individual test articles (BS1, BS2,BS4, 851A, 851B, and 851D) and combinations of test articles (851A and851D; 851B and 851D; and 38K-VZVH and 19H-VZVK), in addition todaratumumab and IgG1 isotype control, were each tested at a final topconcentration of 33 nM, followed by a 7-point five-fold dilution series,in addition to an untreated control, in triplicate. As a positivecontrol for Annexin V staining, cells were treated with 5 mMstaurosporine.

FIG. 18A shows direct apoptosis on Daudi cells for the parentalantibodies (851A, 851B, 851D), two control bispecific antibodies(38K-VZVH, 19H-VZVK), daratumumab and IgG1 isotype control. Daratumumabexhibited the highest level of apoptosis. Both anti-CD19 parents (851A,851B) exhibited a lower level of apoptosis compared to daratumumab. Thetwo bispecific controls and the anti-CD38 parental antibody 851D did notshow appreciable direct apoptosis.

FIG. 18B shows direct apoptosis on Daudi cells for bispecific antibodiesBS1, BS2, BS4, daratumumab and IgG1 isotype control. BS1 and BS2 formatsshowed a significantly higher level of direct apoptosis compared todaratumumab. Bispecific format BS4 showed a level of direct apoptosiscomparable to the parental anti-CD19 851A/851B antibodies (compare FIG.12A); this may be due to the BS4 format not being able to bring the CD19and CD38 into close proximity in order to initiate apoptosis.

FIG. 19A shows cross-linking induced apoptosis on Daudi cells for theparental antibodies (851A, 851B, 851D), two combinations of parentalantibodies (851A+851D; 851B+851D), daratumumab and IgG1 isotype control.Cross-linking increased the level of daratumumab-driven apoptosis(compare FIGS. 12A and 7A). Cross-linking significantly increased thelevel of apoptosis for anti-CD38 851D, which showed no direct apoptosis(compare FIGS. 12A and 7A). The increase in level of apoptosis whencross-linking the anti-CD19 parent antibodies 851A and 851B was lessthan for CD38 antibodies, possibly due to the lower level of CD19,compared to CD38, on Daudi cells (see Table 9). Cross-linkingcombinations of anti-CD19 851A or 851B with anti-CD38 851D did notincrease the level of apoptosis compared to 851D alone.

FIG. 19B shows cross-linking induced apoptosis on Daudi cells forbispecific antibodies BS1, BS2, BS4, (38K-VZVH+19H-VZVK), daratumumaband IgG1 isotype control. When cross-linked, BS1 and B S2 formats showeda level of apoptosis comparable to daratumumab. Notably, bispecificformat BS4 showed a level of cross-linking induced apoptosis comparableto BS1, BS2 and daratumumab; without cross-linking, BS4 showed noapoptosis (see FIG. 6B). The combination of the two control antibodies,38K-VZVH and 19H-VZVK, exhibited significant apoptosis but less than anyof the bispecific formats, showing that including the anti-CD19 andanti-CD38 binding sites in a single antibody is more advantageous thanin independent antibodies.

Example 5 Cytotoxicity

Daudi target cells were treated with a dose response of test articlesand incubated for 15 minutes at 37C/5% CO2. Test articles were tested ata final top concentration of 133 nM, followed by a 7-point five- folddilution series, in addition to 0 nM control. Daratumumab and IgG1isotype control were used as a positive and negative control.

Pre-treated target cells were co-cultured with human PBMCs from n=3donors (E:T 25:1). PBMCs had been “primed” overnight with 100 U/mL ofIL-2. PBMCs were ViaFluor 405-labeled. Samples were incubated for 4hours at 37C/5% CO2 prior to flow cytometry analysis for cytotoxicity.For cytotoxicity analysis, cells were stained with Propidium Iodide(P.I.) and analyzed by high throughput flow cytometry. The percentage ofP.I.+cells within the VF405-population was analyzed as a measure oftarget cell cytotoxicity.

FIGS. 20A, 20B, and 20C show Antibody-Dependent Cellular Cytotoxicity(ADCC) for three donors. For all three donors, the results were similar.The three bispecific formats—BS1, BS2, BS4—and daratumumab exhibitedsimilar levels of ADCC. The anti-CD19 bispecific control 19H-VZVK didnot induce ADCC and was equivalent to the IgG1 control antibody,possibly due to low levels of CD19 on the target Daudi cells (see Table9). In contrast, the anti-CD38 bispecific control 38K-VZVH exhibitedADCC equivalent to the bispecifics and daratumumab, likely due to themuch higher level of CD38 on the Daudi cells compared to CD19.

FIGS. 21A-C show ADCC for three donors. For all three donors, theresults were similar. The three bispecific formats—BS1, BS2,BS4—exhibited similar levels of ADCC. Afucosylated versions of BS1, BS2,BS4 showed increased ADCC of about 10-fold compared to the fucosylatedversions.

Complement-Dependent Cytotoxicity (CDC) assays were also performed.Target cells were treated with a dose response of the following testarticles: BS1, BS2, 38K-VZVH, 19H-VZVH, 38K-VZVH/19H-VZVH combination,as well as controls of Darzalex®, anti-CD20, WT IgG1 Tafasitimab, andhuman IgG1 isotype control. All were tested at a top concentration of133 nM, followed by a five-fold dilution series, 7 points total, inaddition to no treatment controls. After 15 minutes of incubation at37C, 5% CO2, complement was added to treated cells at a finalconcentration of 25%. Cells were then incubated with complement for anadditional 2 hours at 37C, 5% CO2. After complement incubation, cellswere washed and resuspended with 5 ug/mL of a viability dye, propidiumiodide (P.I.), and acquired via high throughput flow cytometry.

FIGS. 22A and 22B show results of complement-dependent cytotoxicity(CDC) assays. The positive technical control, anti-CD20, induced robust,dose-dependent CDC activity. 38K-VZVH and 19H-VZVH (either alone or incombination), anti-CD19 tafasitimab (wt IgG1), and human IgG1 isotypecontrol did not induce any CDC activity. Darzalex®, BS1, and BS2 allshowed CDC activity (though not to the same magnitude as anti-CD20,which is expected from the literature). The maximum cytotoxicity ofDarzalex® was higher than that of both BS1 and BS2.

Antibody-dependent cellular phagocytosis (ADCP)was further assayed bypHrodo' Green AM (pHG) labeled Raji cells treated with a dose responseof test articles and incubated for 15 minutes at 37C, 5% CO2. pHG is apH sensitive dye, only weakly fluorescent at neutral pH, but highlyfluorescent at low pH in the mature phagosomes of macrophages. pHGlabeled Raji target cells with anti-CD20 antibody and IgG1 isotypecontrol were used as a positive control and negative control, with a topconcentration of 133 nM, 7-point five-fold dilution series, and 0 nMcontrol. Pre-treated target cells were co-cultured with humanmacrophages (in vitro differentiated from monocytes) from n=3 donors(E:T 1:2). Macrophages were labeled with Cell Trace' Violet (CTV).Samples were incubated for 4 hours at 37C, 5% CO2 prior to flowcytometry analysis for phagocytosis. The percentage of pHGhi/CTV+ cellswas analyzed as a measure of target cell phagocytosis. Percentages wereplotted on an XY chart against the log of the test articleconcentration, and the data fit to a four-parameter non-linearregression curve from which the EC50 was calculated.

FIG. 23 shows results of antibody-dependent cellular phagocytosis (ADCP)assays using Raji cells as target and donor macrophages. The positivecontrol, anti-CD20, demonstrated dose-dependent phagocytosis for allthree of the donors after 4 hours (between 5-10% max phagocytosis). Thenegative control, IgG1 isotype control, demonstrated no dose-dependentphagocytosis for all three of the donors after 4 hours. Darzalex®demonstrated dose-dependent phagocytosis for all three of the donorsafter 4 hours (between 4-10% max phagocytosis). BS-1, BS-2, afucosylatedBS-1, and afucosylated BS-2 showed slight dose-dependent phagocytosis,with afucosylated formats resulting in an increase in ADCP.

Example 6 Interactions with RBCs

A flow-cytometry based Red Blood Cell (RBC) binding study was performedto evaluate binding of test articles to red blood cells from n=3cynomolgus monkey and n=3 human donors. Whole blood was washed with 1XPBS and then diluted 20-fold with PBS, prior to treatment with testarticles. Bispecifics (BS1, BS2), parental monoclonals (851A, 851D) andcontrols (anti-CD38 Darzalex®, recombinant anti-CD19 tafasitamab, IgG1isotype control, anti-CD47 conjugated to Alexa Fluor™ 647) were testedat a top final concentration of 133 nM followed by a five-fold serialdilution of seven points total, in addition to 0 nM control, intriplicate. Single-arm controls (38K-VZVH, 19H-VZVK) were tested incombination, with both at a top concentration of 133 nM and the samedose response.

After incubation with primary antibodies for 30 minutes on ice, cellswere washed and stained with 5 ug/mL of a secondary antibody (goatanti-human Fcγ F(ab′)2 labeled with Alexa Fluor^(Tm) 647) to detect testarticle binding on red blood cells. Secondary was not used foranti-CD47-A647 stained cells. After incubation with secondary for anadditional 30 minutes on ice, stained cells were washed, diluted, andacquired by high-throughput flow cytometry. The Alexa Fluor™ 647 GeoMeanFluorescence Intensity (MFI) of the single cell population wascalculated. MFI of AF647 was plotted on an XY chart, graphing MFIagainst the log of the concentration, and the data fit to a non-linearregression curve from which the EC50 was calculated.

FIG. 24 shows that AF647-conjugated anti-CD47 showed a dose-responsebinding curve with all three human donors of red blood cells. Darzalex®also showed a dose-dependent increase in binding with all three donors,although the maximum MFI was an order of magnitude less than anti-CD47.Anti-CD38 851D showed the next highest maximum MFI, after Darzalex®,followed by BS1, BS2, 38K-VZVH & 19H-VZVK together, and anti-CD19tafasitamab. Finally, anti-CD19 851A and IgG1 isotype showed only aslight increase in MFI at the highest concentration only.

An in vitro hemagglutination assay was performed on red blood cells froma total of three healthy (n=3) cynomolgus monkey (Cyno) donors and threehealthy (n=3) human donors. Whole blood was acquired the day of thestudy and inspected for coagulation. Blood was then washed with PBS anddiluted 1:50 to obtain the “whole blood substrate”. Whole bloodsubstrate was plated in 96-well round bottom plates and treated withtest articles (BS1, BS2, 38K- VZVH+19H-VZVK, 851A, and 851D), controls(tafasitimab with wild-type IgG1), Darzalex®, and human IgG1 isotypecontrol), or a positive technical control (IGM-55.5), in PBS at a topfinal concentration of 133 nM followed by a five-fold serial dilution ofsix points, in addition to 0 nM control, in triplicate. After 1 hour ofincubation at 37C, 5% CO2, the plate(s) were photographed to ascertainthe level of hemagglutination. Each well was scored on a specifichemagglutination scale from 0-5, using the photographs as a reference.The specific manifestation of each score is somewhat relative to theindividual donor.

FIG. 25A shows results of the hemagglutination assay for human donor 3.The positive control, anti-CD47, induced hemagglutination for all threehuman donors, starting between 0.04 and 1.1 nM. BS1, BS2,38K-VZVH+19H-VZVK, Darzalex®, tafasitimab, and human IgG1 isotypecontrol all showed no induction of hemagglutination at any concentrationfor all three donors. Monoclonal antibodies 851A (anti-CD19) and 851D(anti-CD38) both induced hemagglutination for all three donors, startingat 0.2 or 1.1 nM for each, with a response similar in magnitude to thetechnical control (anti-CD47). In contrast to the parent monoclonalantibodies, BS1 and BS2 did not show any induction of hemagglutinationat any concentration.

FIG. 25B shows results of the hemagglutination assay for cynomolgusdonor 3. The positive control, IGM-55.5 (anti-little i antigen IgMantibody) induced hemagglutination for all three cyno donors starting at0.04 or 0.2 nM. BS1, BS2, 38K-VZVH+19H-VZVK, Darzalex®, tafasitimab, andhuman IgG1 isotype control all showed no induction of hemagglutinationat any concentration for all three donors. Monoclonal antibodies 851A(anti-CD19) and 851D (anti-CD38) both induced hemagglutination for allthree donors, starting at 1.1 nM for each. In contrast to the parentmonoclonal antibodies, BS1 and BS2 did not show any no induction ofhemagglutination at any concentration.

An in vitro hemolysis assay was also performed on red blood cells fromthree (n=3) healthy cynomolgus monkey (cyno) and three (n=3) healthyhuman donors. Whole blood was acquired the day of the study andinspected for coagulation. Blood was washed with PBS and diluted 1:10 toobtain the “whole blood substrate”. The whole blood substrate wastreated with test articles and controls in PBS. Bispecifics (BS1, BS2),parental monoclonals (851A, 851D) and controls (anti-CD38 Darzalex®,recombinant anti-CD19 Tafasitamab, IgG1 isotype control) were tested ata top final concentration of 133 nM followed by a five-fold serialdilution of seven points total, in addition to 0 nM control, intriplicate. Single-arm controls (38K-VZVH,19H-VZVK) were tested incombination, with both at a top concentration of 133 nM and the samedose response. Saponin was tested at a top concentration of 0.1% with athree-fold serial dilution of seven points total. After 1 hour ofincubation at 37C, 5% CO2, plates were centrifuged, and supernatant wascollected. Supernatant was analyzed via plate reader for optical density(OD) at 540 nm. The positive control, Saponin, induced dose-dependenthemolysis starting at 0.001% thru 0.10%, for all species and donors. Notest articles induced any hemolysis at any concentration tested.

FIG. 26 shows that none of the test articles induced any hemolysis atany concentration tested. The positive control, Saponin, induceddose-dependent hemolysis starting at 0.001% thru 0.10%, for all speciesand donors.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

SEQUENCES # SEQUENCE ANNOTATION 1 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAnti-CD19_VH TINWVRQAPGQGLEWMGGIIPIFGIPNYAQKFQGRVTITADESTNTAYMELSSLRAEDTAVYYCA RASGGSADYSYGMDVWGQGTAVTVSS 2DIQMTQSPSSLSASVGDRVTITCRASQGISSWL Anti-CD19_VLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYKRYPYTFGQGTKLEIK 3 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY Anti-CD38_VHAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKF QGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS 4 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAnti-CD38_VL AWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFG QGTKVEIK 5QVQLVQSGAEVKKPGSSVKVSCKASGGTF Anti-CD38_VH ver 2SSYAFSWVRQAPGQGLEWMGRVIPFLGIA NSAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVT VSS 6 QVQLVQSGAEVKKPGSSVKVSCKASGGTFAnti-CD19_Ver2 SSYTINWVRQAPGQGLEWMGGIIPIFGIPNYAQKFQGRVTITADESTNTAYMELSSLRSED TAVYYCARASGGSADYSYGMDVWGQGTL VTVSS 7QVQLVQSGAEVKKPGSSVKVSCKASGGTF Anti-CD19_Ver 3SSYTINWVRQAPGQGLEWMGGIIPIFGIPNY AQKFQGRVTITADESTNTAYMELSSLRSEDTAVYYCARASGGSADYSYGMDVWGGGTL VTVSS 11 GGTFSSYT Anti-CD19_VH_CDR1_IMGT 12SYTIN Anti-CD19_VH_CDR1_Kabat 13 GGTFSSY Anti-CD19_VH_CDR1_Chothia 14SSYTIN Anti-CD19_VH_CDR1_Contact 15 GGTFSSYTIN Anti-CD19_VH_CDR1_AbM 21IIPIFGIP Anti-CD19_VH_CDR2_IMGT 22 GIIPIFGIPNYAQKFQGAnti-CD19_VH_CDR2_Kabat 23 PIFG Anti-CD19_VH_CDR2_Chothia 24WMGGIIPIFGIPN Anti-CD19_VH_CDR2_Contact 25 GIIPIFGIPNAnti-CD19_VH_CDR2_AbM 31 ARASGGSADYSYGMDV Anti-CD19_VH_CDR3_IMGT 32ASGGSADYSYGMDV Anti-CD19_VH_CDR3_Kabat 33 SGGSADYSYGMDAnti-CD19_VH_CDR3_Chothia 34 ARASGGSADYSYGMD Anti-CD19_VH_CDR3_Contact35 ASGGSADYSYGMDV Anti-CD19_VH_CDR3_AbM 41 QGISSWLAAnti-CD19_VL_CDR1_IMGT 42 RASQGISSWLA Anti-CD19_VL_CDR1_Kabat 43 SQGISSWAnti-CD19_VL_CDR1_Chothia 44 SSWLAWY Anti-CD19_VL_CDR1_Contact 45RASQGISSWLA Anti-CD19_VL_CDR1_AbM 51 AAS Anti-CD19_VL_CDR2_IMGT 51AASSLQS Anti-CD19_VL_CDR2_Kabat 53 AAS Anti-CD19_VL_CDR2_Chothia 54SLIYAASSLQ Anti-CD19_VL_CDR2_Contact 55 AASSLQS Anti-CD19_VL_CDR2_AbM 61QQYKRYPYT Anti-CD19_VL_CDR3_IMGT 62 QQYKRYPYT Anti-CD19_VL_CDR3_Kabat 63YKRYPY Anti-CD19_VL_CDR3_Chothia 64 QQYKRYPY Anti-CD19_VL_CDR3_Contact65 QQYKRYPYT Anti-CD19_VL_CDR3_AbM 71 GGTFSSYA Anti-CD38_VH_CDR1_IMGT 72SYAFS Anti-CD38_VH_CDR1_Kabat 73 GGTFSSY Anti-CD38_VH_CDR1_Chothia 74SSYAFS Anti-CD38_VH_CDR1_Contact 75 GGTFSSYAFS Anti-CD38_VH_CDR1_AbM 81VIPFLGIA Anti-CD38_VH_CDR2_IMGT 82 RVIPFLGIANSAQKFQGAnti-CD38_VH_CDR2_Kabat 83 PFLG Anti-CD38_VH_CDR2_Chothia 84WMGRVIPFLGIAN Anti-CD38_VH_CDR2_Contact 85 RVIPFLGIANAnti-CD38_VH_CDR2_AbM 91 ARDDIAALGPFDY Anti-CD38_VH_CDR3_IMGT 92DDIAALGPFDY Anti-CD38_VH_CDR3_Kabat 93 DIAALGPFDAnti-CD38_VH_CDR3_Chothia 94 ARDDIAALGPFD Anti-CD38_VH_CDR3_Contact 95DDIAALGPFDY Anti-CD38_VH_CDR3_AbM 101 QGISSWLA Anti-CD38_VL_CDR1_IMGT102 RASQGISSWLA Anti-CD38_VL_CDR1_Kabat 103 SQGISSWAnti-CD38_VL_CDR1_Chothia 104 SSWLAWY Anti-CD38_VL_CDR1_Contact 105RASQGISSWLA Anti-CD38_VL_CDR1_AbM 111 AAS Anti-CD38_VL_CDR2_IMGT 112AASSLQS Anti-CD38_VL_CDR2_Kabat 113 AAS Anti-CD38_VL_CDR2_Chothia 114SLIYAASSLQ Anti-CD38_VL_CDR2_Contact 115 AASSLQS Anti-CD38_VL_CDR2_AbM121 QQYNSYPRT Anti-CD38_VL_CDR3_IMGT 122 QQYNSYPRTAnti-CD38_VL_CDR3_Kabat 123 YNSYPR Anti-CD38_VH_CDR3_Chothia 124QQYNSYPR Anti-CD38_VH_CDR3_Contact 125 QQYNSYPRT Anti-CD38_VH_CDR3_AbM150 P-X1-L-G-X2-A; wherein X1 and X2 Anti-CD38_VH_CDR2 is any amino acid151 PFLGTA Anti-CD38_VH_CDR2 152 PHLGIA Anti-CD38_VH_CDR2 153 PHLGTAAnti-CD38_VH_CDR2 154 PQLGIA Anti-CD38_VH_CDR2 155 PQLGTAAnti-CD38_VH_CDR2 201 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY BS1-19HTINWVRQAPGQGLEWMGGIIPIFGIPNYAQKFQ GRVTITADESTNTAYMELSSLRAEDTAVYYCARASGGSADYSYGMDVWGQGTAVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 202 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYBS1-38K AFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYC ARDDIAALGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 203QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY BS2-19H1TINWVRQAPGQGLEWMGGIIPIFGIPNYAQKFQ GRVTITADESTNTAYMELSSLRAEDTAVYYCARASGGSADYSYGMDVWGQGTAVTVSSGGGG SGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKRYPYTFGQGTKLEIKAAEPKSSDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 204QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY BS2-19H2TINWVRQAPGQGLEWMGGIIPIFGIPNYAQKFQ GRVTITADESTNTAYMELSSLRAEDTAVYYCARASGGSADYSYGMDVWGQGTAVTVSSGGGG SGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKRYPYTFGQGTKLEIKGGGGDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 205QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY BS2X-19H1AFSWVRQAPGQGLEWMGRVIPFLGIANSAQKF QGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSSGGGGSGG GGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIKAAEPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 206QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY BS2X-19H2AFSWVRQAPGQGLEWMGRVIPFLGIANSAQKF QGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSSGGGGSGG GGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIKGGGGDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 207QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY BS3-19H38TINWVRQAPGQGLEWMGGIIPIFGIPNYAQKFQ GRVTITADESTNTAYMELSSLRAEDTAVYYCARASGGSADYSYGMDVWGQGTAVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GGGGSGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPF LGIANSAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH 208 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS BS3-FcRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 209QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSY BS4-38K19AFSWVRQAPGQGLEWMGRVIPFLGIANSAQKF QGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGG GGSGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTINWVRQAPGQGLEWMGGIIPIFGIP NYAQKFQGRVTITADESTNTAYMELSSLRAEDTAVYYCARASGGSADYSYGMDVWGQGTAVT VSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKS LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKRYPYTFGQGTKLEIK 210 DIQMTQSPSSLSASVGDRVTITCRASQGISSWL 19VL-CLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYKRYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 211DIQMTQSPS SLSASVGDRVTITCRASQGIS SWL 38VL-CLAWYQQKPEKAPKSLIYAAS SLQSGVPSRF SGSG SGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIKRTVAAP SVFIFPP SDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 212QVQLVQSGAEVKKPGSSVKVSCKASGGTF 3C10 scFv-003Fab-Fc KnobSSYTINWVRQAPGQGLEWMGGIIPIFGIPNY (BS3-19F38)AQKFQGRVTITADESTNTAYMELSSLRAED TAVYYCARASGGSADYSYGMDVWGQGTAVTVSSGGGGSGGGGSGGGGSDIQMTQSP SSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYKRYPYTFGQGTKLEIKGGGGGQVQLVQSGAEVKKPGS SVKVSCKASGGTFSSYTINWVRQAPGQGLEWMGGIIPIFGIPNYAQKFQGRVTITADEST NTAYMELSSLRAEDTAVYYCARASGGSADYSYGMDVWGQGTAVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 213DIQMTQSPSSLSASVGDRVTITCRASQGISS 003VL (38VL-CL)WLAWYQQKPEKAPKSLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 214DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Fc Hole (BS3-Fc)MISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 215 QVQLVQSGAEVKKPGSSVKVSCKASGGTF BS1-38K ver2SSYAFSWVRQAPGQGLEWMGRVIPFLGIA NSAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK216 QVQLVQSGAEVKKPGSSVKVSCKASGGTF BS1-19H ver2SSYTINWVRQAPGQGLEWMGGIIPIFGIPNY AQKFQGRVTITADESTNTAYMELSSLRSEDTAVYYCARASGGSADYSYGMDVWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK217 QVQLVQSGAEVKKPGSSVKVSCKASGGTF BS2-19H ver2SSYTINWVRQAPGQGLEWMGGIIPIFGIPNY AQKFQGRVTITADESTNTAYMELSSLRSEDTAVYYCARASGGSADYSYGMDVWGGGTL VTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQ KPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKRYPYTFG QGTKLEIKAAEPKSSDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 218 QVQLVQSGAEVKKPGSSVKVSCKASGGTFBS1M-1 SSYAFSWVRQAPGQGLEWMGRVIPFLGTA NSAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVT VSS 219 QVQLVQSGAEVKKPGSSVKVSCKASGGTFBS1M-3 SSYAFSWVRQAPGQGLEWMGRVIPHLGIA NSAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVT VSS 220 QVQLVQSGAEVKKPGSSVKVSCKASGGTFBS1M-4 SSYAFSWVRQAPGQGLEWMGRVIPHLGTA NSAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVT VSS 221 QVQLVQSGAEVKKPGSSVKVSCKASGGTFBS1M-6 SSYAFSWVRQAPGQGLEWMGRVIPQLGIA NSAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVT VSS 222 QVQLVQSGAEVKKPGSSVKVSCKASGGTFBS1M-7 SSYAFSWVRQAPGQGLEWMGRVIPQLGTA NSAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVT VSS 223 DIQMTQSPSSLSASVGDRVTITCRASQGISSBSM-10 HLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK

1. A method of treating an individual afflicted with a cancer or a tumorcomprising administering to the individual a common light chainbispecific antibody wherein the common light chain bispecific antibodycomprises an anti-human-CD38 immunoglobulin heavy chain variable regionpaired with a common immunoglobulin light chain variable region and ananti-human-CD19 immunoglobulin heavy chain variable region paired withthe common immunoglobulin light chain variable region, wherein theanti-human-CD38 immunoglobulin heavy chain variable region comprises:(a) a heavy chain complementarity determining region 1 (HCDR1)comprising an amino acid sequence set forth in SEQ ID NO: 72; (b) aheavy chain complementarity determining region 2 (HCDR2) comprising anamino acid sequence set forth in SEQ ID NO: 154; and (c) a heavy chaincomplementarity determining region 3 (HCDR3) comprising an amino acidsequence set forth in SEQ ID NO: 93; wherein the anti-human-CD19immunoglobulin heavy chain variable region comprises: (d) a heavy chaincomplementarity determining region 1 (HCDR1) comprising an amino acidsequence set forth in SEQ ID NO: 12; (e) a heavy chain complementaritydetermining region 2 (HCDR2) comprising an amino acid sequence set forthin SEQ ID NO: 23; and (f) a heavy chain complementarity determiningregion 3 (HCDR3) comprising an amino acid sequence set forth in SEQ IDNO: 33; wherein the common immunoglobulin light chain variable regioncomprises: (g) a light chain complementarity determining region 1(LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 103;(h) a light chain complementarity determining region 2 (LCDR2)comprising an amino acid sequence set forth in SEQ ID NO: 113; and (i) alight chain complementarity determining region 3 (LCDR3) comprising anamino acid sequence set forth in SEQ ID NO: 123 thereby treating theindividual afflicted with the cancer or tumor.
 2. The method of claim 1,wherein the anti-human-CD38 immunoglobulin heavy chain variable regioncomprises an amino acid sequence having at least 90% identity to SEQ IDNO: 3 or 5, wherein the anti-human-CD38 immunoglobulin heavy chainvariable region comprises (a), (b), and (c) of claim
 1. 3. The method ofclaim 2, wherein the anti-human-CD38 immunoglobulin heavy chain variableregion comprises an amino acid sequence identical to SEQ ID NO: 3 or 5,wherein the phenylalanine at position 54 of SEQ ID NO: 3 or at position54 of SEQ ID NO: 5 is substituted with glutamine.
 4. The method of claim1, wherein the anti-human-CD19 immunoglobulin heavy chain variableregion comprises an amino acid sequence having at least 90% identity toSEQ ID NO: 1 or 6, wherein the anti-human-CD19 immunoglobulin heavychain variable region comprises (d), (e), and (f) of claim
 1. 5. Themethod of claim 4, wherein the anti-human-CD19 immunoglobulin heavychain variable region comprises an amino acid sequence identical to SEQID NO: 1 or
 6. 6. The method of claim 1, wherein the common light chainbispecific antibody comprises an anti-human-CD38 immunoglobulin heavychain constant region, comprising one or more amino acid substitutionsthat inhibit homodimerization of the anti-human-CD38 immunoglobulinheavy chain constant region and promote heterodimerization of theanti-human-CD38 immunoglobulin heavy chain constant region with anon-anti-human-CD38 immunoglobulin heavy chain constant region.
 7. Themethod of claim 6, wherein the anti-human-CD38 immunoglobulin heavychain constant region comprises a T366W substitution according to EUnumbering or T366S/L368A/Y407V substitution according to EU numbering.8. The method of claim 1, wherein the common light chain bispecificantibody comprises an anti-human-CD19 immunoglobulin heavy chainconstant region comprising one or more amino acid substitutions thatinhibit homodimerization of the anti-human-CD19 immunoglobulin heavychain constant region and promote heterodimerization of theanti-human-CD19 immunoglobulin heavy chain constant region with anon-anti-human CD19 immunoglobulin heavy chain constant region.
 9. Themethod of claim 8, wherein the anti-human-CD19 immunoglobulin heavychain constant region comprises a T366W substitution according to EUnumbering or a T366S/L368A/Y407V substitution according to EU numbering.10. The method of claim 1, wherein the common immunoglobulin light chainvariable region further comprises an immunoglobulin light chain constantregion.
 11. The method of claim 1, comprising an anti-human-CD19immunoglobulin heavy chain comprising the amino acid sequence set forthin SEQ ID NO: 201, a common immunoglobulin light chain comprising theamino acid sequence set forth in SEQ ID NO: 213, and an anti-human-CD38immunoglobulin heavy chain comprising the amino acid sequence set forthin SEQ ID NO: 202 or 215 wherein the phenylalanine at position 54 of SEQID NO: 202 or SEQ ID NO: 215 is substituted with glutamine.
 12. Themethod of claim 1, wherein the anti-human-CD19 immunoglobulin heavychain variable region comprises an A84S or an Al 08L substitutionaccording to Kabat numbering.
 13. The method of claim 1, wherein thecommon immunoglobulin light chain variable region comprises a W32Hsubstitution according to Kabat numbering.
 14. The method of claim 1,wherein the common light chain bispecific antibody is included in acomposition further comprising a pharmaceutically acceptable diluent,carrier, or excipient.
 15. The method of claim 1, wherein the commonimmunoglobulin light chain variable region comprises an amino acidsequence having at least 90% identity to SEQ ID NO: 4, wherein thecommon immunoglobulin light chain variable region comprises (g), (h),and (i) of claim
 1. 16. The method of claim 1, wherein the commonimmunoglobulin light chain variable region comprises the amino acidsequence of SEQ ID NO:
 4. 17. The method of claim 1, wherein the commonlight chain antibody is administered intravenously
 18. The method ofclaim 1, wherein the cancer or the tumor is a hematological cancer. 19.The method of claim 18, wherein the hematological cancer is a B cellmalignancy.
 20. The method of claim 1, wherein the cancer or the tumoris a solid-tissue cancer.
 21. The method of claim 20, wherein thesolid-tissue cancer comprises breast cancer, prostate cancer, pancreaticcancer, lung cancer, kidney cancer, stomach cancer, esophageal cancer,skin cancer, colorectal cancer, brain cancer, or head and neck cancer.